Generation of neural stem cells from undifferentiated human embryonic stem cells

ABSTRACT

The present invention relates to the generation of neural cells from undifferentiated human embryonic stem cells. In particular it relates to directing the differentiation of human ES cells into neural progenitors and neural cells and the production of functioning neural cells and/or neural cells of a specific type. The invention also includes the use of these cells for the treatment of neurological conditions such as Parkinson&#39;s disease.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/360,826 filed on Jan. 30, 2012, which is a divisional of U.S. patentapplication Ser. No. 12/134,521 filed on Jun. 6, 2008, now U.S. Pat. No.8,133,730, which is a divisional of U.S. patent application Ser. No.11/005,518 filed on Dec. 3, 2004, now U.S. Pat. No. 7,604,992, which isa continuation of PCT Application No. PCT/AU03/00704 filed on Jun. 5,2003, which claims the benefit of priority of Australia PatentApplication Nos. 2003902348 filed on May 15, 2003, 2003901537 filed onMar. 28, 2003, 2003901536 filed on Mar. 28, 2003, 2002951874 filed onOct. 4, 2002 and PS2793 filed on Jun. 5, 2002. The contents of all ofthe above applications are incorporated by reference as if fully setforth herein.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 69929SequenceListing.txt, created on 15 May2017, comprising 6,329 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the generation of neural cells fromundifferentiated human embryonic stem cells. In particular it relates todirecting the differentiation of human ES cells into neural progenitorsand neural cells and the production of functioning neural cells and/orneural cells of a specific type. The invention also includes the use ofthese cells for the treatment of neurological conditions such asParkinson's disease.

BACKGROUND OF THE INVENTION

Embryonic stem (ES) cell lines are derived from the pluripotent cells ofthe early embryo. These cell lines, potentially, can maintain a normalkaryotype through an infinite life span in vitro and their pluripotentstem cells can differentiate into any cell type. ES cell lines derivedfrom human blastocysts allow the study of the cellular and molecularbiology of early human development, functional genomics, generation ofdifferentiated cells from the stem cells for use in transplantation ordrug discovery and screening in vitro.

The mammalian nervous system is a derivative of the ectodermal germlayer of the post-implantation embryo. During the process of axisformation, it is thought that inductive signals elaborated by severalregions of the embryo (the anterior visceral endoderm and the earlygastrula organiser) induce the pluripotent cells of the epiblast toassume an anterior neural fate. The molecular identity of the factorselaborated by these issues which direct neurogenesis is unknown, butthere is strong evidence from lower vertebrates that antagonists of theWnt and BMP families of signalling molecules may be involved.

Embryonic stem cells are pluripotent cells which are thought tocorrespond to the epiblast of the pre-implantation embryo. Mouse EScells are able to give rise to neural tissue in vitro eitherspontaneously or during embryoid body formation. The neural tissue oftenforms in these circumstances in amongst a mixture of a range of celltypes.

However, differentiation to a specific neural cell population isrequired to realize many of the potential applications of ES cells inregenerative medicine of the central nervous system and neuroscience.Alteration of the conditions of culture, or subsequent selection ofneural cells from this mixture, has been used in the mouse system toproduce relatively pure populations of neural progenitor cells fromdifferentiating cultures of mouse ES cells. These neural progenitorsgave rise to the neuronal and glial lineages in-vitro. Transplantationexperiments have demonstrated the potential of mouse ES derived neuralcells to participate in brain development and to correct variousdeficits in animal model systems.

Human ES cells have been demonstrated to give rise to neural progenitorcells in vitro and have further demonstrated the capability of theprogenitors to differentiate in vitro into mature neurons. In Reubinoffet al, 2000, 2001 PCT/AU99/00990, PCT/AU01/00278 and PCT/AU01/00735methods are described that allow the derivation of highly enriched(>95%) expandable populations of proliferating neural progenitors fromhuman ES cells. The neural progenitors could be induced to differentiatein vitro into astrocyte, oligodendrocyte and mature neurons.Transplantation experiments demonstrated the potential of the neuralprogenitors to integrate extensively into the developing host mousebrain, to respond to local host cues, and to construct the neuronal andglial lineages in vivo (Reubinoff et al., 2001, PCT/AU01/00278).

To derive the neural progenitors, mixed somatic differentiation wasinduced by prolonged culture of undifferentiated human ES cells withoutreplacement of the mouse embryonic fibroblast feeder layer (Reubinoff etal 2000, 2001 PCT/AU99/00990, PCT/AU01/00278). Under these cultureconditions, distinct areas comprised of small piled, tightly packedcells that do not express markers of undifferentiated ES cells or earlyneuroectodermal progenitors were formed among many other cell types.When these areas were mechanically removed and further cultured indefined media that promote the propagation of neural progenitors, theygave rise to the highly enriched preparations of the neural progenitors.

Recently, others have also reported the derivation of neural progenitorsfrom human ES cells (Zhang et al., 2001, Carpenter et al., 2001).However, these authors induced non-specific mixed differentiation ofhuman ES cells by the formation of embryoid bodies (EBs). Followingplating of the EBs and culture in defined medium supplemented withmitogens, enrichment for neural progenitors was accomplished by cellsorting or selective separation following enzymatic digestion. Directeddifferentiation of human ES cells into neural progenitors and furtherinto specific types of neural cells was not reported by these authors.

Directed differentiation of human ES cells into neural progenitors andfurther on into specific types of neural cells may be highly valuablefor basic and applied studies of CNS development and disease. Controlleddifferentiation of human ES cells into the neural lineage will allowexperimental dissection of the events during early development of thenervous system, and the identification of new genes and polypeptidefactors which may have a therapeutic potential such as induction ofregenerative processes. Additional pharmaceutical applications mayinclude the creation of new assays for toxicology and drug discovery,such as high-throughput screens for neuroprotective compounds.Controlled generation of neural progenitors and specific types ofneurons or glia cells from human ES cells in vitro may serve as anunlimited donor source of cells for tissue reconstruction and for thedelivery and expression of genes in the nervous system.

Directed differentiation of human ES cells into neural progenitors, hasbeen demonstrated with the bone morphogenetic protein antagonist nogginin Pera et al., 2001 and PCT/AU01/00735. Treatment of undifferentiatedhuman ES cell colonies that were cultured on feeders with noggin blockeddifferentiation into extra embryonic endoderm and uniformly directed thedifferentiation into a novel cell type (“noggin cells”). Noggin cellsare similar in terms of morphology and lack of expression of markers ofundifferentiated stem cells or neural progenitors to the small piled,tightly packed cells that were obtained within a mixture of other celltypes in high density cultures.

When noggin cells were transferred to defined culture conditions theygave rise to neural progenitors, neurons and glial cells.

A major application of human ES cells may be their potential to serve asa renewable unlimited donor source of cells for transplantation.However, the potential use of human ES cell derived neural cells inregenerative medicine will depend on their capability to restorefunction. So far the potential of human ES cell derived neural cells torestore function after transplantation has not been demonstrated.

In the mouse, ES cell derived progeny may be functional. Transplantationof low doses of undifferentiated mouse ES cells into the rat striatumresults in their differentiation into dopaminergic neurons andrestoration of cerebral function and behaviour in animal model ofParkinson's disease (Bjorklund et al 2002). Nevertheless, it should benoted that teratoma tumors were observed in 5 of 22 transplanted animalsand in 6 grafted rats no surviving ES cells were found. Teratomaformation and the relatively low survival rate post transplantationpreclude the clinical utilization of this approach.

Parkinson's disease is the second most common neurodegenerative disorderaffecting over one million patients in the USA. Pharmacologicaltreatments of the disorder, mainly with L-dopa, have limited long termsuccess and are associated with serious motor side effects.Transplantation of dopaminergic neurons (DA neurons) is an alternativeapproach that potentially may overcome the drawbacks of pharmacologicaltreatments (Lindvall 1997). Clinical trials of transplantation of fetalderived DA neurons into Parkinson's patients show clinical benefits insome patients (Bjorklund and Lindvall 2000; Freed et al., 2001).Nevertheless, the ethical and practical problems of obtaining sufficientfetal donor tissue severely limit widespread application of this mode oftherapy. In vitro production of transplantable dopaminergic cells at alarge scale could circumvent this drawback. A potential source for theunlimited generation of transplantable dopaminergic neurons in vitro isembryonic stem (ES) cell lines.

The potential of ES cells to serve as an unlimited donor source ofdopaminergic neurons (DA) has been demonstrated in the mouse ES cellsystem (Lee at al 2000, Kawasaki at al 2000).

Furthermore, functional recovery following transplantation of mouse EScell-derived DA neurons into an animal model of Parkinson's disease wasrecently demonstrated (Kim et al., 2002). However, it is known in theart of biology that murine and human ES cells are different in manyaspects. Accordingly, methods that are efficient with mouse ES cells maybe unsuitable for human pluripotent stem cells. For example, thecytokine leukemia inhibitory factor (LIF) can support undifferentiatedproliferation of mouse ES cells (Robertson E 1987) while it has noeffect on human ES cells (Reubinoff et al., 2000).

FGF8 and SHH signals control dopaminergic cell fate in the anteriorneural plate. In the mouse, expansion of mouse ES cell derived neuralprogenitors in the presence of FGF and/or SHH significantly increasesthe generation of DA neurons (Lee et al 2000). The combination of SHHand FGF8 fails to induce significant dopaminergic differentiation ofneurons that are derived from human EC cells (NT2/hNT, Stull andLacovitti 2001). This further enhances the differences between human andmouse. Human EC cells resemble human ES cells (Pera M F 2000), and theirlack of response may suggest that pluripotent stem cells from a humanorigin as opposed to their mouse counterpart do not respond to theSHH/FGF8 combination.

Human ES cells can spontaneously differentiate into tyrosine hydroxylase(TH) producing neurons (PCT/AU01/00278, Reubinoff et al., 2001).However, there has been no demonstrated control for the production ofdopaminergic neurons at high yields from human ES cells and moreimportantly the directed differentiation toward a cell type which hasthe potential for transplantation and treatment of neurologicalconditions.

Accordingly, it would be desirable to direct the differentiation ofhuman ES cells toward a useful cell type and to generate the cell typein high yield to improve the chances of successful transplantation.

Therefore, it is an object to overcome some of these practical problemsand problems of the prior art.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is provided a method ofdirecting the fate of undifferentiated hES cells towards neuralprogenitor cells in vitro said method including the steps of:

culturing undifferentiated human ES cells in a defined serum free mediumthat contains FGF-2 and an antagonist of bone morphogenic proteins(BMP).

In this method, the differentiation of human ES cells is directed into aneural fate and differentiation towards other lineages is eliminated.

In another aspect of the present invention there is provided anon-committed culture of neural progenitor cells prepared by this methodas well as isolated non-committed neural progenitor cell derived fromthis method.

In yet another aspect of the present invention there is provided amethod of directing neural fate in a human embryonic stem (hES) cell invitro said method comprising the steps of:

obtaining a neural progenitor cell from a hES cell culture; and

culturing the neural progenitor cell in the presence of a neural fateinducer selected from the group including at least one of FibroblastGrowth Factor (FGF), Sonic Hedgehog Protein (SHH), cAMP inducers,Protein Kinase C (PKC) inducers, dopamine and ascorbic acid (AA) or anycombination thereof.

The present method provides for a controlled differentiation of neuralprogenitors, preferably towards a transplantable neural cell thatestablishes in a predetermined region of the body. Highly enrichedpreparations of these cells may be obtained by the methods describedherein. The newly derived cells have improved transplantability and aremore potent in vivo.

This improved potency translates to improved survival and/or function ofthe differentiated cells upon transplantation.

In another aspect of the present invention there is provided a method ofdirecting neural fate in a human embryonic stem (hES) cell in vitro saidmethod comprising the steps of:

obtaining a neural progenitor cell from a hES cell culture; and

inducing an overexpression of Nurr 1 and/or Lmx1b in the hES cell.

Without being limited by theory, applicants propose that theoverexpression of the Nurr1 and/or Lmx1b gene can direct thedifferentiation of hES cells toward a neural fate and DA neurons.Applicants have shown that the hES cells that have differentiated towardthe neural fate show an over-expression of the Nurr1 gene. Theexpression is maintained during differentiation into neurons thatco-express Nurr1 and TH.

The Nurr 1 and/or Lmx 1b expression may be induced by any methodsavailable to the skilled addressee. Preferably, the gene(s) areintroduced by genetic modification. The gene(s) may be introduced by asuitable vector under the influence of an inducer such that whendifferentiation is to be effected, expression of the gene may be inducedby introduction of the inducer to the cell culture.

In another aspect of the invention, there is provided a method ofenhancing the survival of transplanted DA neurons said method comprising

obtaining a neural progenitor cell from a hES cell culture or a celldifferentiated from the neural progenitor; and

from the neural progenitor.

Without being limited by theory, Applicants propose that a forcedexpression of GDNF and/or BDNF by the transplanted hES cells or theirneural progeny may enhance the survival of transplanted DA neurones.Preferably the expression is an over-expression above a level that isnaturally present.

The neural progenitors may be according to the neural progenitorsdescribed above. They may be genetically modified to include vectorsthat express GDNF and/or BDNF and which may be under the influence of aninducer that can be switched on at an appropriate time to enhance thesurvival of the transplanted cell.

In yet another aspect of the present invention there is provided agenetically modified hES cell that has been prepared by the methodsdescribed above. Preferably, the cell can differentiate to a glial celland can preferably be directed to differentiate upon forced expressionof the Nurr1 and/or Lmx 1b gene and/or the GDNF and/or BDNF survivalfactors.

The present invention also contemplates transgenic animals having themodified genes.

In a further embodiment, the invention includes methods of treatingneural conditions using the genetically modified hES cell, said methodcomprising transplanting the genetically modified hES cell and inducingthe expression of the Nurr1 and/or Lmx 1b gene and/or the GDNF and/orBDNF survival factors.

The method describes “directing neural fate”. This term as used hereinmeans to guide the differentiation and development of neural progenitorspreferably toward a midbrain fate, or toward neuronal cell types,preferably neurons that show characteristics typical of midbrainneurons. The method may be used to generate any neural progenitor orneuronal subtype including but not limited to hES derived motor neurons,GABAergic, glutamerigic, cholinergic, dopaminergic and serotonergicneurons. The method preferably directs a midbrain neural fate to theneural progenitors derived from hES cells. More preferably the neuralprogenitor cell is committed to a midbrain fate, tyrosine-hydroxylase(TH) positive (TH⁺) cell or doparminergic cell.

In a preferred aspect of the present invention there is provided amethod of directing midbrain fate to a hES cell in vitro, said methodcomprising the steps of:

obtaining a neural progenitor cell from a hES cell culture; and

culturing the neural progenitor cell in the presence of a midbrain fateinducer selected from the group including any one of FGF-1, FGF-8,FGF-17, SHH, AA, cAMP inducers, PKC inducers and dopamine or anycombination thereof.

It is most preferred that the midbrain fate inducer is a combination ofFGF-1, FGF-8, FGF-17, SHH, IBMX, forskolin, PMA/TPA and dopamine.Various other combinations may be useful including the combination of:

(i) FGF-8 and SHH, IBMX, forskolin, PMA/TPA and dopamine; or

(ii) FGF-17 and SHH, IBMX, forskolin, PMA/TPA and dopamine; or

(iii) FGF-1, and IBMX, forskolin, PMA/TPA and dopamine; or

(iv) FGF-8 alone; or

(v) FGF-17 alone; or

(vi) IBMX, forskolin, PMA/TPA, and dopamine.

In a further preferred embodiment, the method further includes culturingthe neural progenitor in the presence of ascorbic acid (AA) or ananalogue thereof.

In an even further preferred embodiment, the method further includesculturing the neural progenitor in the presence of NT4 or equivalentthereof such as NT3.

In yet an even further preferred embodiment, the method includes thefurther step of:

culturing the neural progenitor cells on poly-D-lysine and laminin.

The neural progenitor may also be in the form of neurospheres.

In another aspect of the present invention there is provided a cellculture comprising neural progenitors committed to a neural fate,preferably a midbrain fate. Preferably the neural progenitors are inaggregates or sphere structures.

More preferably when these aggregates are induced to differentiate atleast 30% of them give rise to a significant number (>50) of TH+neurons. The proportion of clumps containing TH+ cells may increase toat least 60% when the midbrain fate of the progenitors is enhanced bymidbrain inducers as detailed above.

Preferably, the neural progenitors with a midbrain fate induced byexposure to midbrain fate inducers express mRNA of key genes in thedevelopment of midbrain and dopaminergic neurons, and give rise to TH+neurons or dopaminergic neurons.

In another aspect of the present invention, there is provided anisolated human neural progenitor cell having a committed neural fatemore preferably a committed midbrain fate. Preferably the cell candifferentiate into a TH+ neuron or a dopaminergic neuron. Mostpreferably, the cell is prepared by methods described herein andisolated from a culture of differentiated hES cells that have beeninduced to differentiate toward a midbrain fate by the use of midbrainfate inducers described herein.

In another aspect of the present invention, there is provided anisolated human neuronal cell having a committed neural fate, morepreferably to a committed midbrain fate. Preferably the cell is TH+neuron or a dopaminergic neuron. Most preferably, the cell is preparedby methods described herein and isolated from a culture ofdifferentiated hES cells that have been induced to differentiate towarda midbrain fate by the use of midbrain fate inducers described herein.

In another aspect of the present invention there is provided a humanneural fate inducer composition for inducing neural fate in a culturedhES cell, said composition comprising a neural fate inducer selectedfrom the group including Fibroblast Growth Factor (FGF), ascorbic acid(AA), Sonic Hedgehog Protein (SHH), cAMP inducers, Protein Kinase C(PKC) inducers and dopamine or any combination thereof.

In yet another aspect of the present invention, there is provided amethod of treating a neurological condition in an animal, said methodcomprising administering an effective amount of an in vitro derivedneural progenitor cell to the animal. Preferably, the neural progenitoris derived from an undifferentiated hES cell and is not committed to aneural fate. More preferably the neural progenitor cells have acommitted neural fate. Most preferably, the neural progenitors arecommitted to a midbrain fate.

The commitment of the neural progenitors to a midbrain fate may bedetermined by demonstrating that the neural progenitors express keygenes in the development of midbrain and dopaminergic neurons in vivo.

In a preferred embodiment, the neurological condition is Parkinson'sdisease.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1B shows dark field images of hES colonies treated with noggintwo weeks after passage. (FIG. 1A) A colony mainly comprised of areas ofsmall tight cells (FIG. 1B) A colony with presumably neural rosettes.

FIGS. 2A-2B shows characterization of cells within areas that arepresumably neural rosettes. Fluorescent images of immunostaining for themarkers (FIG. 2A) N-CAM and (FIG. 2B) nestin. A very high proportion ofthe cells express these markers.

FIGS. 3A-3D shows phase contrast micrograph of neurospheres at varioustimes after derivation. The spheres gradually acquired in culture around uniform appearance. Spheres generated from noggin treated coloniesat 15 (FIG. 3A) and 27 (FIG. 3B) days after derivation. Spheres at 1(FIG. 3C) and 7 (FIG. 3D) days after transfer of hES cell clumps toneural progenitor medium supplemented with noggin.

FIG. 4 shows characterization of the phenotype of cells within spheresthree weeks after transfer of hES cell clumps into neural progenitormedium with or without noggin. The proportion of cells that expressmarkers of neuroectoderm, endoderm and mesoderm was evaluated byimmunostaining. A high percentage of the cells within the spheresexpress early neural markers. Noggin treatment reduced in a dosedependent manner the percentage of cells expressing markers of endodermand mesoderm.

FIG. 5 shows dark field images of clumps of undifferentiated ES cells,and differentiated clumps after culture (3 weeks) in NPM supplementedwith FGF2 and noggin (a), the same medium without noggin (b), andknockout medium (c). Clumps from groups a and b were further cultured 3weeks in NPM supplemented with FGF2 in the absence of noggin.

FIG. 6 shows indirect immunofluorescence analysis of the percentage ofcells expressing neural markers within hES cell clumps after three weeksof culture in KO medium, NPM+FGF2 and NPM+FGF2+noggin.

FIG. 7 shows indirect immunofluorescence analysis of the percentage ofcells expressing neural markers within hES cell clumps that werecultured 3 weeks in NPM+FGF2 or NPM+FGF2+noggin followed by additional 3weeks in NPM+FGF2.

FIG. 8 shows indirect immunofluorescence analysis of the percentage ofcells expressing endodermal (probably extraembryonic) and mesodermalmarkers within hES cell clumps after three weeks of culture in KOmedium, NPM+FGF2 and NPM+FGF2+noggin.

FIG. 9 shows indirect immunofluorescence analysis of the percentage ofcells expressing endodermal (probably extraembryonic) and mesodermalmarkers within hES cell clumps that were cultured 3 weeks in NPM+FGF2with or without noggin followed by additional 3 weeks in NPM+FGF2.

FIGS. 10A-10F shows fluorescent images of differentiated clumps ofneural cells after treatment with FGF8, SHH and AA and plating onlaminin in the presence of AA. A large proportion of the cells expressthe neuronal marker β-tubulin type III (FIG. 10A, FIG. 10D red). Asignificant number of the cells are immunoreactive with anti TH (FIG.10B, FIG. 10E, green). Images of double staining for both markers showthat TH+ cells coexpress β-tubulin type III (FIG. 10C, FIG. 10F, yellow)

FIGS. 11A-11E shows fluorescent images of differentiated clumps ofneural progenitors after treatment with FGF17 and AA. A significantproportion of the cells are immunoreactive with anti TH (FIGS. 11A-11C)while sparse TH+ cells are observed within non-treated lumps (FIG. 11D,FIG. 11E).

FIG. 12 shows the effect of various combinations of external factors onthe proportion of TH+ clumps. Clumps with >50 TH+ cells were scored asTH+ ones. Each bar represents 2-3 experiments. 50-150 clumps were scoredin each experiment.

FIG. 13 shows the effect of treatment with FGF-1, IBMX, forskolin, PMA(TPA), dopamine and AA on the generation of TH+ clumps. Clumps with >50TH+ cells were scored as TH+ ones. Each bar represents the scoring of65-200 clumps.

FIG. 14 shows a confocal microscopy image of double immunostaining forTH (green) and β-tubulin type III (red).

Neurospheres that were propagated for 5 weeks, were plated on lamininand allowed to differentiate for a week in the presence of ascorbicacid. The image is a projection of multiple confocal microscopy imagesof consecutive planes through the differentiating clumps of neuralcells.

FIG. 15 shows the percentage of neurons expressing TH followingtreatment with FGF8 and AA. Neural spheres were plated on laminin in theabsence of mitogens and treated with AA, FGF8 or both AA and FGF8 for aweek. The neural spheres were then further cultured for an additionalweek in the presence of AA. Each bar represents confocal imaginganalysis of 150-300 cell bodies for the expression of TH and β-tubulinIII within 10-15 random fields.

FIG. 16 shows indirect immunofluorescence images of differentiatedneurons decorated with anti TH and anti DAT antibodies.

FIGS. 17A-17B shows indirect immunofluorescence images of adifferentiated neuron coexpressing Nurr1 and TH (FIG. 17A). The neuronwas developed from transduced hES cells over-expressing Nurr1. Schematicpresentation of the lentiviral vector that was used to force theexpression of Nurr1 is presented in FIG. 17B.

FIG. 18 shows indirect immunofluorescent analysis of TH expression inthe 6-OH dopamine lesioned rat striatum and in the intact striatum ofcontralateral side.

FIG. 19 shows the percentage of cells expressing neural progenitormarkers within spheres prior to transplantation. Progenitor cells wereanalyzed by indirect immunofluorescence for the expression of markers12-24 h after disaggregating of spheres and plating on an adhesivesubstrate. 93-94% of the progenitors expressed the early neural markersand 27% expressed the neuronal marker β-tubulin type III. The barsrepresent results from three independent experiments.

FIG. 20 shows RT-PCR analysis of expression of regulatory genes ofdevelopment of ES cells, early CNS, midbrain and dopaminergic neuron byneural progenitors and their differentiated progeny. The symbols + and −indicate whether the PCR reaction was done with or without the additionof reverse transcriptase. HES—mainly undifferentiated hES cell colonies;NPs—neural progenitors after 6 weeks in culture and prior totransplantation.

FIG. 21 shows apomorphine-induced rotational behavior in individual shamoperated and human neurosphere transplanted Parkinsonian rats. Data isgiven as percent change in comparison to each rat rotational behavior at2 weeks after transplantation. At this time point the rats exhibited thefull effect of the 6-hydroxydopamine lesions as determined byapomorphine induced rotational behavior. At 12 weeks, allsham-transplanted rats (Ctrl 1-4) showed no difference in rotationalbehavior as compared to baseline. In 4 out of 5 human neurospheretransplanted rats (ES 2-5) there was a significant decrease inrotational behavior.

FIG. 22 shows apomorphine-induced rotational behaviour in individualsham operated and human neurosphere transplanted Parkinsonian rats. Inthis experiment, neurospheres that were passaged for 5 weeks prior totransplantation were used. Data is given as percent change in comparisonto each rat rotational behaviour at 2 weeks after transplantation. Atthis time point the rats exhibited the full effect of the6-hydroxydopamine lesions as determined by apomorphine inducedrotational behaviour. At 8 weeks, all sham-transplanted rats (Ctrl 1-3)showed no difference in rotational behaviour as compared to baseline. Inall 5 human neurosphere transplanted rats (ES 1-5) there was asignificant decrease in rotational behaviour.

FIG. 23 shows apomorphine-induced rotational behaviour in sham operatedand human neurosphere transplanted Parkinsonian rats. At 2, 4, 8 and 12weeks after transplantation, the severity of the disease was scored andcompared between hES cell-transplanted (♦; n=16) andvehicle-transplanted (□; n=12) animals by quantification of rotationalbehaviour in reaction to apomorphine.

Data is presented as percent change (mean±SEM) in comparison torotational behaviour at 2 weeks after transplantation. At this timepoint, the rats exhibited the full effect of the 6-OH-DA lesions. Asignificant decrease in rotational behaviour was observed intransplanted animals (70% of baseline versus 105% in controls, at 12weeks after transplantation, p<0.05, student t-test).

FIG. 24 shows rotational behaviour in response to amphetamine. Thenumber of amphetamine-induced rotations was significantly lower inneural sphere-transplanted animals (n=11) compared to sham operatedcontrol animals (n=10) (P<0.004, student t-test).

FIG. 25 shows the results of non-pharmacological behavioural evaluationof hES cell-transplanted rats. In both stepping adjustments and forelimbplacing non-pharmacological tests there is a significant increase inmobility after stem cell therapy (p<0.003, student t test).

FIGS. 26A-26B shows fluorescent images of a trail of human cells,identified by a human specific anti-mitochondrial antibody along thetransplantation tract in the Parkinsonian rat striatum, at 24 hour posttransplantation (FIG. 26A) and 1 month post transplantation (FIG. 26B).Arrows indicate areas of recipient striatum, near the transplantationtract, without anti-mitochondria+ cells.

FIG. 27 shows fluorescent images of the stratum and the injection tractsafter immuno-staining for the neural progenitor marker nestin. Many ofthe transplanted human cells are in a progenitor state, as indicated byexpression of the intermediate filament protein nestin.

FIG. 28 shows anti PCNA immunostaining of cells within the neuralprogenitor grafts. At 24 hours after transplantation the majority ofcells expressed PCNA (red), while sporadic expression was observed after12 weeks. Nuclei were stained with DAPI (blue).

FIG. 29A-29E shows fluorescent images of transplanted human cellsexpressing TH and DAT. (FIGS. 29A-29D) Double immuno-fluorescentstaining demonstrating TH+ cells (red) that are immunoreactive with theanti-human specific mitochondria antibodies (green) within the graft ofhuman neural cells 3 months after transplantation. (FIG. 29A) Lowmagnification image demonstrating TH+ cells predominantly in the edgesof the trail of engrafted cells. (FIG. 29B) High magnification image ofthe TH+ cells within the graft (nuclei are counterstained with DAPI((blue). (FIGS. 29C-29D) Confocal microscopy images of single cells thatare co-expressing human mitochondrial antigen (FIG. 29C) and TH (FIG.29D, nuclei are indicated by asterisks). Cells immunoreactive withanti-human DAT (green), within the lesioned striatum, are demonstratedin (FIG. 29E) (nuclei are counterstained with DAPI (blue)).

FIG. 30 shows RT-PCR analysis of expression of human specific midbrainand dopamine neuron markers within brain samples from the area of thegraft. The human specific transcripts were expressed in stem celltransplanted animals (n=2) while they were not detected in a controlvehicle transplanted animal.

FIGS. 31A-31J shows the derivation and characterization of spheres. Darkfield stereo-microscope images of an undifferentiated hES cell colonyone week after passage (FIG. 31A), noggin treated colony at two weeksafter passage (FIG. 31B), and hES cell derived spheres (FIG. 31C).Indirect immunofluorescence staining of the progenitor cells, 12 hoursafter disaggregating of spheres and plating on adhesive substrate, forPSA-NCAM (FIG. 31D), A2B5 (FIG. 31E), N-CAM (FIG. 31F), and nestin (FIG.31G), demonstrated that >90% of the progenitors within the spheresexpressed markers of neural progenitors (FIG. 31J). Followingspontaneous differentiation, 30% of the progenitors differentiated intoneurons and were immunoreactive with β-tubulin III (FIGS. 31J, 31H and31I (red)). Doubleimmunolabeling showed that 0.5% and 1% of the cellsco-expressed β-tubulin III (red) and TH (green, FIG. 31H) or serotonin(green, FIG. 31I).

FIG. 32 shows human ES cell-derived NPs express key regulatory genes ofmidbrain development. Semi-quantitative RT-PCR analysis demonstrated theexpression of transcripts of key regulatory genes in midbrain anddopaminergic neurons development as well as markers of dopaminergicneurons within cultures of both undifferentiated and differentiated NPs.Transcripts of OCT4, which is a marker of undifferentiated hES cellswere not expressed by the NPs. The symbols + and − indicate whether thePCR reaction was done with or without the addition of reversetranscriptase.

FIGS. 33A-33M shows immunohistochemical characterization of transplantedhES-derived neural cells in the brain. Transplanted cells wereidentified by human-specific antibodies. Staining with human specificanti-mitochondria antibody (FIG. 33A, green) showed the linear graftwithin the striatum. The human origin of the graft was confirmed bystaining with a human-specific anti ribonuclear protein antibody (FIG.33B, red), that was localized to the cell nuclei (insert, counterstainwith Dapi in blue). At 24 hr post transplantation there was highexpression of nestin in the graft (FIG. 33C). At 12 weeks posttransplantation there were graft-derived neurons in the rat brains, asindicated by double stainings for human specific markers and neuronalmarkers. Low power field of slides double stained with humanmitochondria (FIG. 33D, green) and neurofilament (FIG. 33D, red) showedthat the majority of transplant did not stain with the neuronal marker.There were some neurofilament+ human cells (FIG. 33D, insert),especially near the interface with the rat brain tissue. Also, therewere human RNP+ cells (FIG. 33E, red) that co-labelled with the neuronalmarker NeuN+ (FIG. 33E, green). The generation of dopaminergic neuronsby the graft was indicated by the presence of TH+ fibers (FIG. 33F, red)within the human mitochondria+ graft (FIG. 33F, green; insert as highpower field). Con-focal microscopy confirmed the presence of humanmitochondria+ cells (FIG. 33G) co-staining for TH (FIG. 33H). Generationof dopaminergic neurons was confirmed by staining with antibodiesdirected against human dopamine transporter (FIG. 33I). Also, there werehuman mitochondria+ cells (FIG. 33J) that co-labelled with V-MAT (FIG.33K). At 24 hours post transplantation the majority of transplantedcells expressed the proliferative marker PCNA (FIG. 33L, in red over ablue Dapi counterstain). At 12 weeks almost no PCNA+ cells were found(FIG. 33M, blue dapi counterstain without red PCNA stain). Space bars:FIGS. 33A-33D, 33F, 50 μm; FIGS. 33E, 33G-33M, 10 μm.

FIG. 34 shows RT-PCR analysis of striata samples from sphere and vehiclegrafted animals for the expression of human-specific transcripts ofmidbrain and dopaminergic neuron markers. The human-specific transcriptswere expressed only by animals (n=3 animals) that received hES-derivedNPs and were not detected in animals that received sham operation (n=2animals). Human-specific primers were used to detect transcripts of En1,En2, TH, AADC and GAPDH. The β-actin primers were not human specific.The symbols + and − indicate whether the PCR reaction was done with orwithout the addition of reverse transcriptase.

FIGS. 35A-35D shows transplantation of hES cell-derived neural spheresimproves motor function in Parkinsonian rats. The number ofd-amphetamine or apomorphine-induced rotations was calculatedindividually for each rat as percentage of its performance at baseline.For each time point the value represents the mean±SE percent rotations.Rotational behaviour that was induced by d-amphetamine (FIG. 35A) andapomorphine (FIG. 35B) decreased significantly in transplanted animalsas compared to baseline and to control rats. *p<0.05 as compared tobaseline and to controls for the pharmacological tests and to thecontrol group for the non-pharmacological tests. Stepping (FIG. 35C,p=0.0012) and placing (FIG. 35D, p=0.0003) also improved significantlyin transplanted rats as compared to controls.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

In a first aspect of the present invention there is provided a method ofdirecting the fate of human embryonic stem cells towards neuralprogenitor cells in vitro said method including the steps of:

culturing undifferentiated human ES cells in a defined serum free mediumthat contains FGF-2 and an antagonist of bone morphogenic proteins(BMP).

In this method, the differentiation of human ES cells is directed into aneural progenitor and differentiation towards other lineages issubstantially eliminated. These neural progenitors are not committed atthis stage and have the potential to become committed if placed underconditions to induce commitment.

The direction of the differentiation is influenced by the use of thedefined serum free media in particular by the presence of FGF-2 and theBMP antagonist. The present invention provides a method of inducingneural progenitor cells from undifferentiated hES cells such that theprocess that directs neural progenitor cells is augmented toward aneural lineage whereas other lineages are eliminated or less prominent.

Preferably the BMP antagonist is selected from the group including adirect antagonist such as fetuin, noggin, chordin, gremlin, follistatin,Cerberus, amnionless, DAN or the ectodomain of BMRIA (a BMP receptorprotein), or ligand binding domains from other BMP receptors. Preferablythe BMP antagonist is noggin.

As previously described in the applicants own applicationPCT/AU01/00735, noggin used in combination with hES stem cells producesa neural progenitor culture. However, the culture is a mixture of neuralprogenitors and other cell types. This aspect of the present inventionrefines the differentiation to neural progenitors and neural cell typesby the use of defined medium conditions which preferably includes theuse of FGF-2 with or without noggin.

In another aspect of the present invention there is provided a cellculture comprising neural progenitor cells differentiated from hES cellsand wherein the neural progenitors are non-committed to a neural fate.These cultures have the potential to commit to a neural fate.

In another aspect of the present invention, there is provided anisolated neural progenitor cell differentiated from a hES cell andwherein said neural progenitor is not committed to a neural fate. Thiscell type has the potential to commit to a neural fate.

The neural progenitor cells prepared by this process may benon-committed neural progenitors that are not committed to anyparticular type of neural cell such as but not limited to neuronal andglial cell types. These cells may be used, as described below andinduced to commit to a neural fate and neuronal cell type preferablyincluding a midbrain cell type. Preferably these cells have a potentialto commit to a neural fate.

The characteristics and phenotype of cells following differentiation andpropagation in culture may be analysed for the expression of the earlyneural markers such as, but not limited to nestin, A2B5, N-CAM, PSA-NCAMand β-tubulin III. The analysis may be conducted at a suitable time tomonitor the progression of the cells through the differentiationprocess. The cells may be analysed after 3 to 6 weeks of culture in NPMsupplemented with FGF2.

The percentage of cells expressing early neural markers increases overtime, preferably over three weeks in culture in NPM+FGF2 in comparisonto KO medium. Noggin treatment further significantly increases thepercentage of cells expressing the neural markers. Preferably the earlyneural markers including nestin, PSA-NCAM, A2B5 and NCAM are increased.Preferably at least 75% of the cells in any culture expresses the earlyneural cell markers after culture with an antagonist of bone morphogenicproteins (BMP). Preferably the antagonist of bone morphogenic proteins(BMP) is noggin. Preferably, at least 95 to 100% of the cells show anincrease in expression of the neural markers.

More preferably, A2B5 expression is increased to at least about 95% ofthe cells in culture; and NCAM expression is increased to at least about73% of the cells in culture, more preferably the expression is increasedto at least about 90%. After an additional 3 weeks of culture inNPM+FGF2 the percentage of cells expressing most of the neural markersmay stabilize. The major effect of an additional 3 week culture periodmay increase the percentage of cells expressing NCAM in the noggintreated clumps.

These cells also show a reduction in the expression of non-neuralmarkers such as but not limited to the endodermal marker alpha-fetalprotein, the endodermal marker HNF3α or the epidermal marker keratin-14.Other markers showing reduction in expression include laminin and lowmolecular weight cytokeratin; muscle actin, smooth muscle actin anddesmin

At the RNA level, RT-PCR analysis may be used to confirm that in thenoggin treated clumps the expression of the endodermal markeralpha-fetal protein, the endodermal marker HNF3α or the epidermal markerkeratin-14 is reduced. Preferably these markers are significantlyreduced at approximately 3 weeks and preferably undetectable at 6 weeks.

The cells may also be distinguished by their overexpression of Nurr-1.More preferably the cells co-express Nurr-1 and TH. The expression ofNurr-1 may be maintained during differentiation into neuronsparticularly those co-expressing Nurr-1 and TH.

Preferably, the neural progenitors are obtained from undifferentiatedhES cells that are directed to differentiate into neural cells byculture in suspension preferably as clumps in defined serum free culturemedium preferably neural progenitor media (NPM) in the absence offeeders. The NPM may be supplemented with FGF-2, with/without EGF and/orLIF.

The NPM may contain DMEM/F12 (1:1), B27 supplementation (1:50),glutamine 2 mM, penicillin 50 u/ml and streptomycin 50 μg/ml (Gibco),and supplemented with 20 ng/ml fibroblast growth factor 2 (FGF2) with orwithout 20 ng/ml human recombinant epidermal growth factor (EGF), and 10ng/ml human recombinant LIF (R & D Systems, Inc., Minneapolis, Minn.).

The cells may be cultured in suspension and may be exposed to noggin inthe range of 350-700 ng/ml.

FGF-2 is used to promote proliferation and prevent the differentiationof the undifferentiated non-committed hES cell derived neuralprogenitors in the presence of noggin. A suitable FGF-2 concentration isapproximately 20 ng/ml.

In another aspect of the present invention, there is provided a culturedundifferentiated ES cell which is committed to differentiate to a neuralprogenitor.

In another aspect of the present invention there is provided a method ofdirecting neural fate in a human embryonic stem (hES) cell in vitro saidmethod comprising the steps of:

obtaining a neural progenitor cell from a hES cell culture; and

culturing the neural progenitor cell in the presence of a neural fateinducer selected from the group including at least one of FibroblastGrowth Factor (FGF), Sonic Hedgehog Protein (SHH), cAMP inducers,Protein Kinase C (PKC) inducers, dopamine and ascorbic acid (AA) or anycombination thereof.

The present method provides for a controlled differentiation of neuralprogenitors, preferably towards a transplantable neural cell thatestablishes in a predetermined region of the body. Highly enrichedpreparations of these cells may be obtained by the methods describedherein. The newly derived cells have improved transplantability and aremore potent in vivo. This improved potency translates to improvedsurvival and/or function of the differentiated cells upontransplantation.

The method describes “directing neural fate”. This term as used hereinmeans to guide the differentiation and development of neural progenitorspreferably toward a midbrain fate, or toward neuronal cell types,preferably neurons that show characteristics typical of midbrainneurons. The method may be used to generate any neural progenitor orneuronal subtype including but not limited to hES derived GABAergic,glutamerigic, cholinergic, motor neurons, dopaminergic and serotonergicneurons. The method preferably directs a midbrain neural fate to theneural progenitors derived from hES cells.

More preferably the neural cell is a neural progenitor cell committed toa midbrain fate, tyrosine-hydroxylase (TH) positive (TH⁺) cell ordopaminergic cell.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises”, is not intended to exclude other additives, components,integers or steps.

The neural progenitor cells may be obtained by any means that providesthese cells from a culture of hES cells preferably an undifferentiatedculture of hES cells. The hES cells may be spontaneously differentiatedor induced to differentiate preferably as described in the applicant'sown applications namely PCT/AU01/00278 and PCT/AU01/00735, the contentsof which are incorporated herein.

Preferably, the neural progenitor cells are non-committed hES cellderived neural progenitors that have not been committed to anyparticular neural cell type or fate.

In a further preferred embodiment, the neural progenitor cells areobtained herein from a hES cell culture treated with noggin or otherinhibitors or antagonists of bone morphogenic proteins (BMP). The use ofnoggin directs the undifferentiated hES cell within colonies that arecultured on feeders into areas comprised of small fight cells and areaswith neural rosettes. Dissection of these areas and transferal intodefined serum free culture conditions may produce characteristicpreparations of proliferating neural progenitors. The serum free cultureconditions may include media which may be neural progenitor growthmedium (NPM) supplemented with fibroblast growth factor (FGF) preferablyFGF-2, with/without epidermal growth factor (EGF) and/or leukaemiainhibitory factor (LIF) A typical NPM may contain DMEM/F12 (1:1), B27supplementation (1:50), glutamine 2 mM, penicillin 50 u/ml andstreptomycin 50 μg/ml (Gibco), and supplemented with 20 ng/ml humanrecombinant epidermal growth factor (EGF), and 20 ng/ml fibroblastgrowth factor 2 (FGF2) (R & D Systems, Inc., Minneapolis, Minn.).

In a further preferred embodiment the neural progenitors are obtainedfrom undifferentiated hES cells that are directed to differentiate intoneural cells by culture in suspension preferably as clumps in definedserum free culture medium preferably NPM in the absence of feeders. TheNPM may be supplemented with FGF-2, with/without EGF and/or LIF.

In an even further preferred embodiment the undifferentiated hES cellsthat are cultured in suspension as above, are also treated with noggin.Noggin may be used in a range of 350-700 ng/ml. The addition of nogginfurther promotes the differentiation towards the neural lineage and intoneural progenitors, while it reduces the differentiation into non-neurallineages. These cultures provide neurospheres comprising neuralprogenitors that may be used to differentiate toward a neural cell linewith a committed neural fate when cultured in the presence of the neuralfate inducers.

During the culturing of the hES cells and differentiation towards neuralprogenitors, these cells may be cultured in the presence of FGF,with/without EGF and/or LIF. Preferably the FGF is FGF-2. A suitableconcentration of FGF-2 is approximately 20 ng/ml. EGF may be added inthe form of naturally produced or recombinantly produced EGF, morepreferably human EGF is more suitable for hES cells. A suitableconcentration for EGF is approximately 20 ng/ml. LIF may be added topromote proliferation of neural progenitors during induction ofdifferentiation when noggin is presented to the cells simultaneously.Under these conditions NPM may be supplemented with FGF-2 and LIF. LIFis preferably a human LIF and suitably used in a concentration ofapproximately 10 ng/ml.

In another aspect of the invention there is provided a method to blockdifferentiation of hES cells towards non-neural lineages. Exposure ofhES cells cultured in NPM to noggin blocks the differentiation tonon-neural lineages, preferably mesoderm, endoderm (probablyextraembryonic) and epidermal lineages.

Additional culture of the spheres in NPM+FGF2 without noggin furthereliminates non neural cells, preferably endodermal (probablyextraembryonic) epidermal and mesodermal cells. This is evidenced by thereduction of the expression of non-neural markers.

In another aspect of the present invention there is provided a method ofdirecting neural fate in a human embryonic stem (hES) cell in vitro saidmethod comprising the steps of:

obtaining a neural progenitor cell from a hES cell culture; and

inducing an overexpression of Nurr 1 and/or Lmx1b in the hES cell.

Without being limited by theory, applicants propose that theoverexpression of the Nurr1 and/or Lmx1b gene can direct thedifferentiation of hES cells toward a neural fate and DA neurons.Applicants have shown that the hES cells that have differentiated towardthe neural fate show an over-expression of the Nurr1 gene. Theexpression is maintained during differentiation into neurons thatco-express Nurr1 and TH.

The Nurr 1 and/or Lmx 1b expression may be induced by any methodsavailable to the skilled addressee. Preferably, the gene(s) areintroduced by genetic modification. The gene(s) may be introduced by asuitable vector under the influence of an inducer such that whendifferentiation is to be effected, expression of the gene may be inducedby introduction of the inducer to the cell culture. Preferably the cellsare genetically modified using the lentiviral vector transduction systemas described in PCT/AU02/0175.

In another aspect of the invention, there is provided a method ofenhancing the survival of transplanted DA neurons said method comprising

obtaining a neural progenitor cell from a hES cell culture;

inducing an expression of GDNF and/or BDNF in the neural progenitor cellor a cell differentiated from the neural progenitor.

Without being limited by theory, Applicants propose that a forcedexpression of GDNF and/or BDNF by the transplanted hES cells or theirneural progeny may enhance the survival of transplanted DA neurones.Preferably the expression is an over-expression above a level that isnaturally present.

The neural progenitors may be according to the neural progenitorsdescribed above. They may be genetically modified to include vectorsthat express GDNF and/or BDNF and which may be under the influence of aninducer that can be switched on at an appropriate time to enhance thesurvival of the transplanted cell. The timing may coincide with a periodthat improves the survival of the cell. Preferably, the GDNF and/or BDNFis induced when the cells are transplanted. However, these factors maybe induced during the differentiation stage to enhance their survival.

Differentiated and transplantable hES cells of the present invention maybe modified to express Nurr1 and/or Lmx 1b along with GDNF and/or BDNFto provide enhanced survival of transplanted hES cells.

Preferably, the cells differentiate to glial cells. Preferentially, thecells are transplanted as hES cells capable of differentiation and thedifferentiation is induced in vivo in the presence of the induced genesNurr1 and/or Lmx 1b. Further induction of the survival factors GDNFand/or BDNF may also be present in vivo.

In yet another aspect of the present invention there is provided agenetically modified hES cell that has been prepared by the methodsdescribed above. Preferably, the cell can differentiate to a glial celland can preferably be directed to differentiate upon forced expressionof the Nurr1 and/or Lmx 1b gene and/or the GDNF and/or BDNF survivalfactors.

The present invention also contemplates transgenic animals having themodified genes.

In a further embodiment, the invention includes methods of treatingneural conditions using the genetically modified hES cell, said methodcomprising transplanting the genetically modified hES cell and inducingthe expression of the Nurr1 and/or Lmx 1b gene and/or the GDNF and/orBDNF survival factors.

The neural progenitor cells are cultured in the presence of neural fateinducers to induce them to differentiate toward a specific neuralprogenitor cell preferably committed to a midbrain fate or a neural orneuronal cell type preferably with a committed midbrain fate.

The term “neural fate inducer” is any substance that can direct theneural progenitor toward a neural cell type such as, but not limited toa progenitor of a specific neural fate such as but not limited tomidbrain fate, midbrain neurons and any neuronal cell type selected fromthe group including hES derived GABAergic, glutamerigic, cholinergic,dopaminergic, serotonergic and motor neurons. The substance(s) alsopromotes survival of neurons such as to promote growth, function,augment activity of functioning cells, enhance synthesis ofneurotransmitter substances, enhance activity of naturally occurringnerve growth promoting factors, prevent degeneration of neurons, induceregrowth whilst directing the cell toward a neural fate and enhancingsurvival of the differentiated neural cell.

The term “FGF” as used herein may include, but is not limited to, FGF-1,FGF-2, FGF-6, FGF-8, FGF-9, FGF-98 and FGF-17, or any biologicallyactive fragment or mutein thereof. Preferably for the induction of humanneurons from hES cells, it is preferable to use FGF-1, FGF-8 or FGF-17alone or in combination. The FGF may derive from any animal, preferablymammalian, more preferably human. Natural or recombinantly produced FGF,preferably FGF-1, FGF-8 or FGF-17 may be used.

Biologically active variants of FGF are also encompassed by the methodof the present invention. Such variants should retain FGF activities,particularly the ability to bind to FGF receptor sites.

FGF activity may be measured using standard FGF bioassays, which areknown radioreceptor assays using membranes, a bioassay that measures theability of the molecule to enhance incorporation of tritiated thymidine,in a dose-dependent manner, into the DNA of cells, and the like.Preferably, the variant has at least the same activity as the nativemolecule.

The biologically active variants can be FGF analogues or derivatives.The term “analogue” as used herein is an analogue of either FGF or anFGF fragment that includes a native FGF sequence and structure havingone or more amino acid substitutions, insertions, or deletions.Analogues having one or more peptoid sequences (peptide mimic sequences)are also included. The term “derivative” as used herein is any suitablemodification of FGF, FGF fragments, or their respective analogues, suchas glycosylation, phosphorylation, or other additions of foreignmoieties, so long as the FGF activity is retained. Methods for makingFGF fragments, analogues, and derivatives are available in the art.

In addition to the above described FGFs, the method of the presentinvention can also employ an active mutein or variant thereof. By theterm active mutein, as used in conjunction with an FGF, is meant toinclude a mutated form of the naturally occurring FGF. FGF muteins orvariants will generally have at least 70%, preferably 80%, morepreferably 85%, even more preferably 90% to 95% or more, and mostpreferably 98% or more amino acid sequence identity to the amino acidsequence of the reference FGF molecule. A mutein or variant may, forexample, differ by as few as 1 to 10 amino acid residues, such as 6-10,as few as 4, 3, 2 or even 1 amino acid residue providing the FGFactivity is maintained.

A new member of the FGF family, FGF 17, was recently discovered(Hoshikawa et al., 1998) Like FGF8 it is predominantly expressed in thedeveloping CNS in the midline region of the forebrain and themidbrain-hindbrain junction. In addition it is expressed in additionaldistinct expression domains (Xu et al., 1999, Heikinheimo et al., 1994,Crossley et al., 1995). FGF-8 is expressed earlier then FGF-17, whoseexpression persists a little longer (Xu et al., 2000). While thesefactors may have a functional relationship in patterning some areas ofthe brain, the role of FGF-17 in CNS development or its effect on EScell differentiation is unknown.

“Sonic Hedgehog Protein” (SHH) refers to any sonic hedgehog proteinderived from any animal, and functional fragments thereof.

“cAMP Inducers” as used herein, may be selected from any compound thatinduces cAMP activity either directly by forskolin or NPA(R(−)-propylnorapomorphine a D2 receptor agonist of PKA, increases cAMP)or indirectly by inhibiting phosphodiesterase byIsobutyl-methoylxanthine (IBMX) or by compounds with IBMX like activitysuch as cAMP-specific Ro 20-1724, Rolipram, or Etazolate but morepreferably selected from the group including Isobutyl-methoylxanthine(IBMX), or forskolin used alone or in combination.

“Protein Kinase C (PKC) Inducers” as used herein may be phorbolmyristate acetate or phorobol 12-myristate 13-acetate which is aspecific activator of PKC group A (α,βI,βII,χ) and PKC group B (δ,ε,η,θ)or tumor promoter activity (TPA). However, any substance that inducesisozyme specific PKC either directly or indirectly is included in thescope of the present invention.

“Dopamine” as used herein includes naturally or synthetically produceddopamine or any functional equivalent or analogue thereof. “Functionalequivalents or analogues” are those compounds that have the sameactivity as naturally produced dopamine that is produced by the adrenalmedulla.

Dopamine, along with epinephrine, norepinephrine, and serotonin, belongsto a chemical family referred to “monoamines”. Within the family ofmonoamines, epinephrine, norepinephrine, and dopamine are derived fromthe amino acid tyrosine and form a subfamily called the catecholamines.Frequently, tyrosine hydroxylase (TH), the rate-limiting enzyme for thebiosynthesis of dopamine, is used as a marker to identify dopaminergicneurons.

In a preferred aspect of the present invention there is provided amethod of directing midbrain fate to a hES cell in vitro, said methodcomprising the steps of:

obtaining a neural progenitor cell from a hES cell culture; and

culturing the neural progenitor cell in the presence of a midbrain fateinducer selected from the group including any one of FGF-1, FGF-8,FGF-17, SHH, AA, cAMP inducers, PKC inducers and dopamine or anycombination thereof.

The neural progenitor may be presented as clumps or clusters of cells,neurospheres or single cells. Clumps or clusters may comprise anynumbers of cells preferably providing the cells can be exposed to theneural fate inducers. However, cells on the outer of the clumps may beinduced toward a neural fate. Preferably the clumps or neurospherescomprise up to 3000 cells per clump, more preferably the clumps comprise2000-3000 cells per clump. The neural progenitors may be presented tothe inducers either in suspension or adherent cultures, preferably thecells are adherent to poly-D-lysine and laminin.

The use of the specific midbrain fate inducers causes thedifferentiation to be directed to midbrain cells rather than neuralcells of other brain regions.

The neural cells may be cultured in the presence of FGF-2 with orwithout EGF and/or LIF. When culturing in the presence of the midbrainfate inducers, FGF-2 and EGF and/or LIF may be removed from the mediumso that the progenitors are more readily directed to take a midbrainfate by treatment with the midbrain fate inducers.

FGF selected from the group including FGF-1, FGF-8 and FGF-17 is used asa midbrain fate inducer. These factors are preferably used incombination with other midbrain fate inducers at a concentration ofapproximately 100-200 ng/ml.

SHH is preferably used in combination with other midbrain inducers at aconcentration of about 0.5-1 μg/ml.

cAMP inducers such as IBMX or forskolin may be used at a concentrationof about 0.25 mM and 50 μM respectively in combination with othermidbrain inducers.

The PKC inducers PMA/TPA may be used in the concentration of 200 nM anddopamine may be used at a concentration of about 20 μM in combinationwith other midbrain inducers.

It is most preferred that the midbrain fate inducer is a combination ofFGF-1, FGF-8, FGF-17, SHH, IBMX, forskolin, PMA/TPA and dopamine.Various other combinations may be useful including the combination of:

(i) FGF-8 and SHH, IBMX, forskolin, PMA/TPA and dopamine; or

(ii) FGF-17 and SHH, IBMX, forskolin, PMA/TPA and dopamine; or

(iii) FGF-1, and IBMX, forskolin, PMA/TPA and dopamine; or

(iv) FGF-8 alone; or

(v) FGF-17 alone; or

(vi) IBMX, forskolin, PMA/TPA, and dopamine.

Preferably, FGF-8 and FGF-1 are used in a concentration of about 200ng/ml.

In a further preferred embodiment, the method further includes culturingthe neural progenitor in the presence of ascorbic acid (AA) or ananalogue thereof.

The addition of AA or an analogue thereof to the cells in the presenceof the midbrain fate inducers improves the survival of the cells andfurther directs the differentiation toward TH⁺ cells. Furthermore, THproduction/expression may be increased. AA or an analogue thereof may beadded together with or following exposure of the cells to midbrain fateinducers. Addition of AA or an analogue thereof at the time of removalof FGF-2 and EGF may improve TH⁺ generation. The AA or an analoguethereof is supplemented to the medium, preferably NPM in the presence orabsence of FGF-2 and EGF and/or LIF. AA or an analogue thereof may beused at a concentration of approximately 400-800 μM.

AA or an analogue thereof may be used with any one or combination ofFGF-1, FGF-8 or FGF-17 with or without other midbrain inducers.

In an even further preferred embodiment, the method further includesculturing the neural progenitor in the presence of NT4 or equivalentthereof such as NT3.

The term “equivalent thereof” as used herein means a sequence ormolecule which functions in a similar way but may have deletions,additions or substitutions that do not substantially change the activityor function of the sequence or molecule.

NT4 is a survival factor like AA. If added at the stage of AA addition,the proportion of TH⁺ neurons can be increased. NT4 may be used at aconcentration of about 20 ng/ml.

In yet an even further preferred embodiment, the method includes thefurther step of:

culturing the neural progenitor cells on poly-D-lysine and laminin.

This additional step will induce further differentiation into neurons.The neural progenitors, having been exposed to midbrain inducers, AAwith or without NT4 may be disaggregated at this stage and plated onpoly-D-lysine and laminin. Generally, the concentration of poly-D-lysineis in the range of about 5 to 15 μg/ml preferably 10 μg/ml and lamininis in the range of about 1 to 10 μg/ml, preferably, 4 μg/ml.

The cells may continue to be cultured in NPM supplemented with AA withor without NT4.

Progression of differentiation throughout the process of the method fromundifferentiated ES cells to uncommitted neural progenitors, committedneural progenitors, and specific types of differentiated neural cellsmay be followed by monitoring the expression of marker genes of variouscell types or key genes in the development in vivo of undifferentiatedES cells, differentiated cells from various lineages, the CNS, specificareas of the CNS, and various types of differentiated neural cells. Theexpression of key genes may be monitored at the protein or mRNA level.RT-PCR, semi-quantitative RT-PCR, real time RT-PCR micro and macroarrays or any other method may be used to monitor the expression ofmRNA.

Preferably, total RNA is extracted from undifferentiated hES cells;differentiated hES cells; neurospheres at various time points alongpropagation and following differentiation. RT-PCR is then used tomonitor the expression of key genes and markers including:transcriptional markers for undifferentiated hES cells (Oct4); markersof endoderm probably of extra embryonic origin (αFP and HNF3α); mesodermmarker (CD34); epidermal marker (keratin 14). Early CNS (central nervoussystem) marker (Otx2); Mesencephalic markers (Pax5, Pax2, wnt1);midbrain markers (Nurr1, Lmx1b, En1. and En2) and markers of thedopaminergic pathway (AADC, TH, Ptx3).

Progression of differentiation throughout the process of the method maybe monitored also by physical assessment of morphology (ascertained bythe trained eye) or by analysis of marker expression at the proteinlevel. Early expression neural markers such as but not limited to N-CAM,A2B5, PSA-NCAM and nestin can help to assess progression toward neuralprogenitors. The markers β-tubulin III, light chain neuro filaments areexpressed by early neurons while heavy chain neurofilaments, MAP-2ab,synaptophysin and neurotransmitors are expressed by mature neurons.

Additionally, measurement of TH⁺ may serve as a marker to identifydopaminergic neurons. Other markers of dopaminergic neurons arearomatic-L-amino acid decarboxylase (AADC) and dopamine transporter(DAT). Dopaminergic neurons as opposed to norepinephric neurons lack theexpression of dopamine β hydroxylase (DBH). The production and secretionof dopamine (measured by RP-HPLC) is a definitive marker of dopaminergicneurons. Electrophysiological methods may be further used tocharacterize the maturity and function of TH+ neurons.

In another aspect of the present invention there is provided a cellculture comprising neural progenitors with a committed fate, preferablya midbrain fate. Preferably the neural progenitors are in aggregates orsphere structures. More preferably when these aggregates are induced todifferentiate at least 30% of them give rise to a significant number(>50) of TH+ neurons. The proportion of clumps containing TH+ cells mayincrease to at least 60% when the midbrain fate of the progenitors isenhanced by midbrain inducers as detailed above.

In a preferred aspect the cell culture of committed neural progenitorscapable of specific neural fate are generated from undifferentiated hESderived neural progenitor ells cultured under defined culture conditionsof defined media.

The neural progenitors that are differentiated from undifferentiated hEScells following noggin treatment and specific culture conditions havethe potential to give rise to multiple lineages but they also have thepotential to further differentiate to cells having a neural fate,preferably a midbrain fate.

The present invention generates cultures of neural progenitors andisolated neural progenitors with a neural fate, preferably a midbrainfate. The neural fate inducers (except the survival factor AA) areremoved from the medium at the time of differentiation from progenitorsinto neurons and still TH+ neurons are obtained. This indicates that theprogenitors are committed to a midbrain fate and will give rise to TH+neurons in the absence of neural fate or midbrain fate inducers. Withoutbeing limited by theory, it is considered that the production ofprogenitors committed to a midbrain fate is important sincetransplantation of committed progenitors may be more effective thandifferentiated neurons since they may have a better survival potentialafter transplantation and may have a higher potential to integrate andinteract with the host brain. The committed progenitors may beidentified by their potential to differentiate into TH+ neurons withoutany treatment with midbrain inducers.

In another preferred aspect of the present invention there is provided acell culture comprising clumps of differentiated hES cells and whereinat least 30% of the clumps include a significant number (>50) of neuronswith a midbrain fate. The proportion of clumps containing TH+ cells mayincrease to at least 60%.

In another preferred aspect of the invention there is provided a cellculture comprising clumps of differentiated hES cells and wherein atleast 30% of the neurons (β-tubulin III+ cells) are expressing TH. Theproportion of neurons expressing TH may increase to at least 60%.

In a further preferred aspect there is provided a cell culturecomprising a population of differentiated hES cells wherein thepopulation is substantially neural progenitors having a midbrain fate.More preferably, the population comprises neural progenitors that cangive rise upon differentiation to neurons that are TH⁺ or dopaminergic.Most preferably, the cell culture is prepared by the methods describedherein.

In an even further preferred aspect there is provided a cell culturecomprising a population of differentiated hES cells wherein thepopulation is substantially neurons having a midbrain fate. Morepreferably, the population comprises neurons that are TH⁺ ordopaminergic. Most preferably, the cell culture is prepared by themethods described herein.

Preferably, the neural progenitors with a midbrain fate induced byexposure to midbrain fate inducers give rise to TH⁺ neurons ordopaminergic neurons. Preferably the cells have improved transplantability and function in-vivo wherein improvement is over differentiatedhES cells that have differentiated spontaneously into non-committedneural progenitor cells.

Preferably, the cells are functional in vivo and more preferably thecells are functional in vivo and have the ability to transplant andrepopulate by proliferation and differentiation.

By “functional” it is meant to include that the neurons can show nervegrowth, be active by enhancing neurotransmitters and by synapticallyactive and influence motor, sensor cognitive autonomous or any othertype of behavior that results from nerve function.

In another aspect of the present invention, there is provided anisolated human neural progenitor cell having a committed neural fate,more preferably a committed midbrain fate. Preferably the cell candifferentiate into a TH⁺ neuron or a dopaminergic neuron. Mostpreferably, the cell is prepared by methods described herein andisolated from a culture of differentiated hES cells that have beeninduced to differentiate toward a midbrain fate by the use of midbrainfate inducers described herein.

In another aspect of the present invention, there is provided anisolated human neuronal cell having a committed neural fate, morepreferably a committed midbrain fate. Preferably the cell is TH⁺ neuronor a dopaminergic neuron. Most preferably, the cell is prepared bymethods described herein and isolated from a culture of differentiatedhES cells that have been induced to differentiate toward a midbrain fateby the use of midbrain fate inducers described herein.

In another aspect of the present invention there is provided a humanneural fate inducer for inducing neural fate in a cultured hES cell andblocking non-neural lineages. The inducer is selected from the group ofBMP antagonists including but not limited to fetuin, noggin, chordin,gremlin, follistatin, Cerberus, amnionless, DAN or the ectodomain ofBMRIA (a BMP receptor protein), or ligand binding domains from other BMPreceptors more preferably the inducer is noggin.

In another aspect of the present invention there is provided a humanneural fate inducer composition for inducing neural fate in a culturedhES cell, said composition comprising a neural fate inducer selectedfrom the group including Fibroblast Growth Factor (FGF), ascorbic acid(AA), Sonic Hedgehog Protein (SHH), cAMP inducers, Protein Kinase C(PKC) inducers and dopamine or any combination thereof.

Preferably the composition is a human midbrain neural fate inducer, morepreferably the composition is a human midbrain neural progenitor inducermore preferably a TH⁺ cell inducer, and even more preferably a dopamineproducing neuron inducer.

Preferably the FGF is selected from the group including FGF-1, FGF-8 orFGF-17.

Preferably the “cAMP Inducers” are selected from any compound thatinduces cAMP activity either directly by forskolin or NPA(R(−)-propylnorapomorphine, a D2 receptor agonist of PKA, increasescAMP) or indirectly by inhibiting phosphodiesterase IBMX like activityby cAMP-specific Ro 20-1724, Rolipram, Etazolate but more preferablyselected from the group including Isobutyl-methoylxanthine (IBMX), orforskolin used alone or in combination.

The “Protein Kinase C (PKC) Inducers” may be phorbol myristale acetate(PMA Phorobol 12-myristate 13-acetate is a specific activator of PKCgroup A α,βI,βII,χ) and PKC group B (δ,ε,η,θ)) or tumor promoteractivity. However, any substance that induces isozyme specific PKCeither directly or indirectly is included in the scope of the presentinvention.

“Dopamine” may include naturally or synthetically produced dopamine orany functional equivalent or analogue thereof.

In yet another aspect of the present invention, there is provided amethod of treating a neurological condition in an animal, said methodcomprising administering an effective amount of in vitro derived neuralprogenitor cell to the animal.

Preferably, the cells are neural progenitors that have the potential togive rise to multiple lineages and have been derived fromundifferentiated hES cell. Preferably, the neural progenitors arecommitted to a neural fate, more preferably, a midbrain fate. Mostpreferably, the neural progenitors have been derived from the methodsdescribed herein.

The present method can be employed to deliver agents or neural cells tothe brain for diagnosis, treatment or prevention of disorders ordiseases of the CNS, brain, and/or spinal cord and or peripheral and orautonomic nervous system. These disorders can be neurologic orpsychiatric disorders. These disorders or diseases include brain diseasesuch as Alzheimer's disease, Parkinson's disease, Lewy body dementia,multiple sclerosis, epilepsy, cerebellar ataxia, progressivesupranuclear palsy, multi-system atrophies, spino-cerebelardegenerations, optic nerve and retinal diseases including retinal andmacular degeneration and retinitis pigmentosa, amyotrophic lateralsclerosis, affective disorders, anxiety disorders, obsessive compulsivedisorders, personality disorders, attention deficit disorder, attentiondeficit hyperactivity disorder, Tourette Syndrome, Tay Sachs, NiemanPick, and other lipid storage and genetic brain diseases and/orschizophrenia. The method can also be employed in subjects sufferingfrom or at risk for nerve damage from cerebrovascular disorders such asstroke in the brain or spinal cord, from CNS infections includingmeningitis and HIV from tumors of the brain and spinal cord, or from aprion disease. The method can also be employed to deliver agents tocounter CNS disorders resulting from ordinary aging (eg anosmia or lossof the general chemical sense), brain injury, or spinal cord injury. Themethod can also be employed to treat diseases of the peripheral andautonomic nervous systems including but not limited to hereditaryneuropathies, inflammatory neuropathies and traumatic neuropathies.

The present method can be employed to deliver agents to the brain fordiagnosis, treatment or prevention of neurodegenerative disorders bygenetically modifying the hES cells.

The term “treatment” is used in its most broadest sense to includeprophylatic (ie preventative) treatment as well as treatments designedto ameliorate the effects of the neurological condition.

An “effective amount” of agent or of hES cells or neural progenitors isan amount sufficient to prevent, treat, reduce and/or ameliorate thesymptoms and/or underlying causes of any of the above disorders ordiseases. In some instances, an “effective amount” is sufficient toeliminate the symptoms of these disease and, perhaps, overcome thedisease itself. When applied to cells, ie effective amount is an amountsufficient to generate cells to prevent, treat, reduce or ameliorate thesymptoms.

The in vitro hES derived neural cells or neural progenitors are thosecells that are either non-committed but are inclined to differentiatetoward a neural progenitor cell type or have an induced committed neuralfate, preferably neural progenitors more preferably neural progenitorswith a committed midbrain fate, or differentiated TH⁺ or dopaminergicneurons. The cells may be identified by neuronal markers such as DAT,RMP and VMAT2. These markers may be identified in the progenitors or thetransplanted cells which have been transplanted. Most preferably, thesecells are produced by the methods described herein.

In a preferred embodiment, the neurological condition is Parkinson'sdisease. Parkinson's disease (PD) is characterized by the progressiveloss in function of dopaminergic neurons. The progressive loss ofdopaminergic function interferes with the normal working of the neuronalcircuitry necessary for motor control so that patients with PD showcharacteristic motor disturbances such as akinesia, rigidity and resttremor. Other symptoms include pain, impaired olfaction, alterations ofpersonality and depression.

According to the invention, neural progenitors or midbrain committedneural progenitors or dopaminergic neuronal cells are generated usingthe cell culturing methods described above. These cells are thenadministered to the brain of the patient in need thereof to producedopamine and restore behavioural deficits in the patient. Preferably,the cells are administered to the basal ganglia of the patient.

The principal therapeutic target in the brain for Parkinson's is thebasal ganglia. Other potential sites are substantia nigra which extendsforward over the dorsal surface of the basis peduncle from the rostralborder of the pans toward the subthalamic nucleus. In additiontherapeutic target areas are also the locus ceruleus which is located inthe rostral pons region and the ventral tegmental area which is locateddorsomedial to the substantia nigra.

According to the invention, the cells are administered to the patient'sor animal's brain. The cells may be implanted within the parenchyma ofthe brain, as well as in spaces containing cerebrospinal fluids, such asthe sub-arachnoid space or ventricles. The cells may be also implantedinto sites outside the central nervous system such as but not limited tothe peripheral and autonomic nerve and ganglia. “Central nervous system”is meant to include all structures within the dura mater.

Typically, the neural cells are administered by injection into the brainof the patient. Injections can generally be made with a sterilizedsyringe. The exact size needle will depend on the species being treated,the needle should not be bigger than 1 mm diameter in any species. Thoseof skill in the art are familiar with techniques for administering cellsto the brain of a patient.

After the neural cells including but not limited to non-committed neuralprogenitors, committed neural progenitors, neuronal cells of varioustypes are formed according to the cell culturing method previouslydescribed, the cells are suspended in a physiologically compatiblecarrier. As used herein, the term “physiologically compatible carrier”refers to a carrier that is compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Those of skillin the art are familiar with physiologically compatible carriers.Examples of suitable carriers include cell culture medium (eg, Eagle'sminimal essential media), phosphate buffered saline, and Hank's balancedsalt solution +/− glucose (HBSS).

The volume of cell suspension administered to a patient will varydepending on the site of implantation, treatment goal and amount ofcells in solution. Typically the amount of cells administered to apatient will be a “therapeutically effective amount”. As used herein, atherapeutically effective amount refers to the number of transplantedcells which are required to effect treatment of the particular disorder.For example, where the treatment is for Parkinson's disease,transplantation of a therapeutically effective amount of cells willtypically produce a reduction in the amount and/or severity of thesymptoms associated with that disorder, eg, rigidity, akinesia and gaitdisorder.

It is estimated that a severe Parkinson's patient will need at leastabout 100,000 surviving dopamine cells per grafted side to have asubstantial beneficial effect from the transplantation. As cell survivalis low in brain tissue transplantional in general (5-10%) an estimated1-4 million dopaminergic neurons should be transplanted. It is estimatedthat a lower number of neural progenitors committed to give rise todopaminergic neurons will be required to produce a similar therapeuticresponse.

Examples of the procedures used in the present invention will now bemore fully described. It should be understood, however, that thefollowing description is illustrative only and should not be taken inany way as a restriction on the generality of the invention describedabove.

EXAMPLES Example 1 Directed Differentiation of hes Cells into HighlyEnriched Cultures of Neural Progenitors

A. Induction of Differentiation on Feeders with Noggin Coupled withManipulation of Culture Conditions.

To derive enriched preparations of neural progenitors, differentiationof human ES cells was directed into neural fate by transfer ofundifferentiated hES cell clumps onto fresh feeders and culture in amodified hES cell medium with reduced serum concentration and in thepresence of the BMP antagonist noggin. Specifically, human ES cells(HES-1 cell line, Reubinoff et al 2000, PCT/AU99/00990) with a stablenormal (46XX) karyotype were cultured on mitomycin C mitoticallyinactivated mouse embryonic fibroblast feeder layer in gelatine coatedtissue culture dishes as previously described (Reubinoff et al., 2000PCT/AU99/00990 and PCT/AU01/00278). To induce differentiation, at theusual passage, clumps of undifferentiated ES cells were plated on freshfeeders and cultured in the usual hES medium supplemented with 10% serum(instead of 20%) and 500 ng/ml of noggin (R&D systems). Noggin (500ng/ml) was further added to the medium every other day throughout a 6-8day culture period (3-4 administrations). After 6-8 days, noggin wasomitted and the cells were further cultured in the modified medium with10% serum for additional 4-6 days. At this time about 12-14 days, about70% of the colonies differentiated mostly into areas that were comprisedof tightly packed small cells with a uniform grey opaque appearanceunder dark field stereo microscope (FIG. 1a , PCT/AU01/00278,PCT/AU01/00735). Other colonies differentiated into areas withstructures that could resemble primitive neural rosettes (FIG. 1b ).

Clumps of about 150 cells were mechanically isolated by using therazor-sharp edge of a micro glass pipette or a razor blade from the greyopaque areas and replated in serum-free medium supplemented with humanrecombinant FGF-2 and EGF. Specifically, the clusters of cells weretransferred to plastic tissue culture dishes containing neuralprogenitors growth medium (NPM) that consisted of DMEM/F12 (1:1), B27supplementation (1:50), glutamine 2 mM, penicillin 50 u/ml andstreptomycin 50 μg/ml (Gibco), and supplemented with 20 ng/ml humanrecombinant epidermal growth factor (EGF), and 20 ng/ml fibroblastgrowth factor 2 (FGF2) (R & D Systems, Inc., Minneapolis, Minn.). Underthese culture conditions the clumps formed free floating sphericalstructures within 24 hours and sequential propagation and expansion ofthe sphere cultures was possible as previously described (Reubinoff atal., 2001, PCT/AU01/00278) for prolonged periods (6 months).

During the initial weeks in culture, the spheres gradually acquired auniform morphology (FIG. 3 A,B). A detailed analysis of markerexpression by the cell within the spheres was conducted at 3-4, 8 and 12weeks after derivation. The spheres were disaggregated into single cellsthat were plated, fixed and analysed by fluorescent immunohistochemistryfor the expression of the early neural markers N-CAM, A2B5, PSA-NCAM andnestin. A high proportion of the cells expressed these markers (N-CAM83±4% A2B5 84±4%, nestin 69±6% PSA-NCAM 74±2%) and the level ofexpression was stable during the 12 weeks period.

The phenotype of cells within the areas with structures that couldresemble primitive neural rosettes (FIG. 1b ) was also analyzed. Theseareas were mechanically dissected, and disaggregated. The cells wereplated on laminin and cultured in NPM for two days. Immunophenotyping atthat point revealed that 95-100% of the cells expressed N-CAM (FIG. 2A)and nestin (FIG. 2B).

B. Induction of Differentiation by Culture of es Cell Aggregates inSerum Free Conditions in the Presence of Noggin and fgf2.

In an alternative approach, undifferentiated human ES cell clumps ofabout 200 cells were transferred into NPM and cultured in suspension inthe presence of noggin (350 and 700 ng/ml) for approximately 3 weeks. Tofurther induce neural differentiation, proliferation and prevent thedifferentiation of neural progenitors the medium was also supplementedwith human recombinant FGF-2 (20 ng/ml). LIF (10 ng/ml) was alsoincluded in the medium in early experiments. The media was replacedtwice a week. Under these culture conditions the clumps of hES cellsturned into round spheres within 7 days (FIG. 3 C,D).

To evaluate the effect of these culture conditions, we have evaluatedthe phenotype of cells within clumps following three weeks of culture.We have examined two concentrations of noggin (350 and 700 ng/ml) incomparison to culture in NPM supplemented with FGF2 in the absence ofnoggin. The clusters were disaggregated into single cells or smallclumps that were plated, cultured for 4 hours on laminin, fixed andanalysed by indirect immunofluorescence for the expression of the earlyneural markers nestin, A2B5 and N-CAM. Markers of endoderm (probablyextraembryonic; low molecular weight (LMW) cytokeratin and laminin) andmesoderm (muscle specific actin and desmin) were examined after a weekof differentiation. Immunofluorescence methods and source of antibodiesare described below in section (C).

A high proportion (^(˜)75%) of the cells within the spheres expressedearly neural markers. The percentage of cells expressing nestin and A2B5was slightly higher in the noggin (700 ng/ml) treated spheres. Noggintreatment had a profound significant effect on the level of expressionof endodermal and mesodermal markers. The proportion of cells expressingendodermal and mesodermal markers was significantly reduced after noggintreatment in a dose dependent manner (FIG. 4).

To analyse and differentiate between the effect of NPM supplemented withFGF2 and noggin on the differentiation of the hES cell clusters we havecharacterized and compared the differentiation of these clustersfollowing culture in three different media (FIG. 5): (a) NPMsupplemented with b-FGF and noggin (700 ng/ml), (b) the same mediumwithout noggin and (c) knockout (KO) medium. After three weeks ofculture a significant difference in the morphology of the clusters wasobserved. Clusters that were cultured in KO medium had the typicalmorphology of embryoid bodies (EBs, FIG. 5c ). Clusters that werecultured in NPM and FGF2 were characterized by cystic structures andareas of dense cells resembling neural spheres (FIG. 5b ). Clusters thatwere cultured in the presence of noggin had the typical morphology ofneural spheres without cystic structures (FIG. 5a ).

Clusters that were cultured in NPM with (FIG. 5a ) or without noggin(FIG. 5b ) were further cultured for an additional 3 weeks in NPMsupplemented with FGF2 in the absence of noggin (FIG. 5).

To characterize and compare the phenotype of cells within the clustersfollowing differentiation and propagation at the various cultureconditions (FIG. 5), the clusters were disaggregated after 3 weeks inculture into single cells or small clumps that were plated, cultured for4 hours on laminin, fixed and analysed for the expression of the earlyneural markers nestin, A2B5, N-CAM, PSA-NCAM and (3-tubulin III (FIG.6). The same analysis was done after additional 3 weeks of culture inNPM supplemented with FGF2 (FIG. 7).

The percentage of cells expressing early neural markers was increasedafter three weeks culture in NPM+FGF2 in comparison to KO medium. Noggintreatment further significantly increased the percentage of cellsexpressing the neural markers (95.6% A2B5; 73% NCAM; FIG. 6). Afteradditional 3 weeks of culture in NPM+FGF2 the percentage of cellsexpressing most of the neural markers was stable in both study groups.The major effect of the additional 3 week culture period was an increasein the percentage of cells expressing NCAM in the noggin treated clumps(from 73% to 93%).

The percentage of cells within the clumps that expressed non-neuralmarkers at 3 weeks (FIGS. 8) and 6 weeks (FIG. 9) of the same floatingcultures as above was analysed. Indirect immuno fluorescence analysis ofthe expression of non-neural markers was performed followingdisaggregation of the clumps and one week of differentiation on laminin.The analysis showed a significant reduction in the percentage of cellsexpressing these markers in the noggin treated cultures after 3 weeks(FIG. 8). Cells expressing these markers were undetectable or rare afteradditional 3 weeks of culture in NPM+FGF2 (FIG. 9).

At the RNA level, RT-PCR analysis confirmed that in the noggin treatedclumps the expression of the endodermal (probably extraembryonic) markeralpha-fetal protein was reduced/undetectable at 3 weeks. This marker wasalso undetectable at 6 weeks. The expression of the endodermal (probablyextraembryonic) marker HNF3α was also reduced/undetectable at 6 weeks ofculture. The expression of transcripts of the epidermal markerkeratin-14 was significantly reduced at 3 weeks and undetectable at 6weeks.

Collectively these data suggest that highly enriched cultures of neuralprogenitors are obtained when undifferentiated clumps of hES cells arecultured in NPM+FGF2 supplemented with noggin. The percentage of neuralprogenitors was higher after differentiation in NPM+FGF2 compared to KOmedium. This is in line with our previous report (PCT/AU01/00278).Noggin treatment further significantly increased the process ofneuralization and blocked the differentiation to extraembryonic endoderm(as previously described in PCT/AU01/00735) and epidermis. Either nogginor the culture conditions (NPM) or both reduced the differentiationand/or did not promote the survival of mesodermal cells.

C. Immunohistochemistry Studies

In general, for the immunophenotyping of disaggregated neural progenitorcells and differentiated neurons, fixation with 4% paraformaldehyde for20 minutes at room temperature was used unless otherwise specified. Itwas followed by blocking with 5% heat inactivated goat serum (Dako) andpermeabilization with 0.2% Triton X (Sigma) in PBS with 0.1% BSA for 30minutes. Samples were incubated with the primary antibodies at roomtemperature for one hour. Cells were washed three times with PBS with0.1% BSA, incubated with the secondary antibodies for 30-45 minutes,counterstained and mounted with Vectashield mounting solution with DAPI(Vector Laboratories, Burlingame, Calif.). Primary antibodieslocalisation was performed by using mouse anti rabbit IgG and goat antimouse IgG conjugated to Cy3 or FITC from Jackson Lab. West Grove, (PA:1:100-1:500), or anti rabbit FITC and goat anti mouse FITC from Dako(1:20-50). Proper controls for primary and secondary antibodies revealedneither non-specific staining nor antibody cross reactivity.

To characterize the immunophenotype of cells within the aggregates,spheres were mechanically disaggregated into single cells or smallclumps and plated on poly-D-lysine and laminin in NPM. The cells werefixed after 1 (for analysis of the expression of neural markers) or—3-7days (for non-neural markers) and examined for the expression of thefollowing markers: laminin (Sigma mouse monoclonal 1:500) and lowmolecular weight cytokeratin ((cytokeratin 8, Beckon Dickinson, SanJose, Calif. ready to use) as markers of endoderm; muscle actin(Reubinoff et al., 2000), smooth muscle actin (Dako mouse IgG 1:50) anddesmin (Dako mouse clone D33 1:50) for mesoderm; nestin (rabbitantiserum a kind gift of Dr. Ron McKay; 1:25 or from Chemicon rabbitanti human 1:100-200), N-CAM (Dako, Carpinteria, Calif.; mouse IgG1:10-20), A2B5 (ATCC, Manassas, Va.; mouse clone105 1:10-20) andPSA-N-CAM (Developmental Studies Hybridoma Bank, mouse undiluted) asmarkers of neural progenitors; β-tubulin III (Sigma; mouse IgG 1:2000)for early neurons.

Two hundred cells were scored within random fields (at ×200 and ×400)for the expression of each of these markers and the experiments wererepeated at least 3-5 times.

D. Marker Characterization by rt-per

RT-PCR was performed as previously described (Reubinoff et al., 2001).Primer sequences (forward and reverse) and the length of amplifiedproducts were as previously described (Reubinoff et al 2001) foralpha-feto-protein and HNF3a and as follows for keratin 8:

(SEQ ID NO: 1) ATGATTGGCAGCGTGGAG, (SEQ ID NO: 2)GTCCAGCTGTGAAGTGCTTG (390 bp).″

Example 2 Directed Differentiation of hES Cell-Derived NeuralProgenitors Towards a Midbrain Fate

Neurospheres that were generated from noggin treated ES cell colonies asdescribed above (Example 1) were propagated 21-35 days in NPMsupplemented with FGF-2 and EGF. At this point the spheres were choppedinto small clumps (2000-3000 cells per clump). FGF-2 and EGF wereremoved from the medium and the progenitors were directed to take amidbrain fate by treatment with various factors for 6-8 days. Duringtreatment with the various factors the clumps were either cultured insuspension or plated on poly-D-lysine and laminin. The factors included.FGF1, FGF8, FGF17 (R&D Systems) at concentrations of 100-200 ng/ml SHH(R&D Systems) at 0.5-1 μg/ml. Other signal transduction inducers: cAMPinducers: IBMX (Sigma, 0.25 mM), forskolin (Sigma 50 μM), PKC inducersPMA (Sigma 200 nM) and dopamine (Sigma 20 μM) were also used. The mediumwas also supplemented with the survival factors Ascorbic acid (AA)(Sigma) at concentration of 400-800 μM and NT4 (R&D Systems) atconcentration of 20 ng/ml. The medium was changed every other day. Toinduce further differentiation into neurons, the spheres were againdisaggregated into small clumps and plated on poly-D-lysine (30-70 kDa.10 μg/ml, Sigma) and laminin (4 μg/ml, Sigma) and cultured in NPM in theabsence of the factors and in the presence of AA with or without NT4 for5-10 days. Differentiated cells were analysed by indirectimmunofluorescence (as described in example 1) for the expression ofβ-tubulin type III (Sigma, 1:1000-3000) Tyrosine hydroxylase (TH) (Sigmaanti mouse monoclonal, 1:250-500 and Pel-Freez anti rabbit polyclonal,1:50-100) dopamine transporter (DAT;_Chemicon; rabbit polyclobal1:50-100) and Nurr1 (Santa Cruz, Calif.; rabbit poly clonal 1:100-200).The proportion of clumps of cells that were comprised of a significantnumber of TH+ cells (>50 cells) was scored. Double immunostaining for THand β-tubulin type III was used to analyse the percentage of cellswithin clumps expressing TH from the total number of neurons β-tubulintype III+cells). Multiple confocal microscopy images of consecutiveplanes through the clump were projected into one image for this analysis(FIG. 14) Ten-fifteen random fields were analysed.

The proportion of cells that differentiated into TH+ neurons when themedium was not supplemented with midbrain fate inducers or survivalfactors was very poor (about 1%, PCT/AU01/00278, FIG. 11D, E) and TH+clumps were not generated. The differentiation into TH+ neurons waspoorer with high passage spheres as opposed to low passage ones andtherefore low passage (30-35 days in culture) spheres were used in thein vitro studies).

Supplementation of the culture medium with ascorbic acid at the time ofremoval of FGF-2 and EGF as well as during differentiation of theprogenitors on substrate gave rise to a significant number of TH+neurons in 35% of the clumps (FIGS. 12 and 13 bar 6) At the cellularlevel, 45% of the neurons in these cultures expressed TH (FIG. 14, 15).

Treatment with FGF-8 (200 ng/ml) in combination with AA significantlyincreased the proportion of TH+ clumps to a level of 67%. (FIG. 10, 12).Analysis at the cellular level, using confocal microscopy, also showedan increase (to 77%) in the percentage of TH+ neurons from the totalnumber of neurons (FIG. 15). In contrast SHH treatment had no effect onthe proportion of TH+ clumps. The effect of SHH was evaluated incombination with AA or in combination with FGF-8 and AA and in bothcases it did not increase the proportion of TH+ neurons (FIG. 12).

To demonstrate that the TH+ neuron enriched cultures that are generatedfollowing treatment with FGF8 and AA include dopaminergic neurons wehave examined the expression of DAT. In vertebrates, DAT is exclusivelyexpressed in DA neurons (Lee et al., 2000). Indirect immunofluorescencestudies demonstrated cells expressing DAT within the cultures ofdifferentiated TH+ cells (FIG. 16). This data suggested that thetreatment with FGF8 and AA induced differentiation into dopaminergicneurons.

FGF-17 was also found to have the potential to induce differentiationtowards a midbrain fate. Following treatment with the combination ofFGF-17 and AA 60% of the clumps were comprised of a significant numberof TH+ neurons (FIG. 12). The inductive effect of FGF-17 was notsignificantly different from the effect of FGF-8. Fluorescent images ofclumps of differentiated neurons following treatment with FGF-17 and AAare demonstrated in FIG. 11. A significant proportion of the cellswithin these clumps express TH.

Directing the differentiation of hES cell derived neural progenitorstowards TH+ neurons was also accomplished by using the combination ofFGF-1, cAMP activators IBMX and Forskolin, Protein kinase C (PKC)activator PMA, dopamine and AA. Following treatment with this set offactors (FGF-1 at 200 ng/ml) 66-75% of the clumps of differentiatedneurons contained a significant number of TH+ neurons (FIG. 13). The setof these factors was as efficient as FGF-8 and AA or FGF-17 and AA. Itshould be noted that a 55% proportion of TH+ clumps was obtained whenthe signal transduction activators (IBMX, Forskolin, PMA) dopamine andAA were used without FGF-1. In contrast to the effect of FGF-1 at 200ng/ml, the addition of FGF-1 or FGF-8 at a concentration of 10 ng/ml tothe set of these factors did not increase the proportion of TH+ clumps(FIG. 13). In these experiments the surviving factor NT4 was added to AAat the stage of final differentiation when the neurospheres were platedon laminin and cultured in NPM.

The transcriptional factor Nurr1 is required for the induction ofmidbrain DA neurons, which fail to develop in Nurr1-null mutant mice(Zetterstrom et al. 1997). Forced expression of the Nurr1 gene may beused to direct the differentiation of human ES cell-derived neuralprogenitors into DA neurons. We have developed a lentiviral vectortransduction system for the introduction of stable genetic modificationsinto human ES cells (Gropp et al., 2003, patent application No.PCT/AU02/0175).

We have used this system to generate hES cells expressing Nurr1. Thevector that we have used (pSIN18.cPPThEF-1α.Nurr1.hPGK.Puro.WPRE)include the mouse Nurr1 gene under the control of hEF1 α promoterfollowed by a selection marker gene (puromycine resistance element)under the control of hPGK promoter. Neural spheres were developed fromthe genetically modified hES cells, propagated and induced todifferentiate in the presence of AA as detailed above. Indirectimmunofluorescence analysis demonstrated neurons coexpressing Nurr1 andTH (FIG. 17). It should be noted that it was not possible to demonstrateby immunostaining the expression of Nurr1 by wild type hES cellsfollowing induction of differentiation according to the same protocol.

Example 3 Improvement of Behavioural Deficit in an Animal Model ofParkinson's Disease Following Transplantation of hES Cell Derived NeuralProgenitors

A Parkinson's disease model in rats was induced by stereotaxic injectionof the neurotoxin 6-hydroxydopamine to cause unilateral nigrostriatallesions. 8 μg/rat of 6-OH dopamine were injected in 4 μl into the rightSubstantia Nigra. Coordinate of injection were P=4.8, L=1.7, H=−8.6.

Two weeks after the injection of neurotoxin the disease severity wasexamined in each rat individually by administration of apomorphine (25μg/100 g body weight) and quantification of contralateral rotationalbehaviour by computerized rotameter system (San-Diego Instruments). Itshould be noted that in early experiments, rotations were counted by anobserver 4 times every 12 minutes, for three minutes each time afterapomorphine administration. Animals with strong baseline rotationalbehavior (>500 rotations/hour) were selected for transplantation. Inthese animals immunofluorescent studies of brain sections demonstratedthat the injection of the neurotoxin resulted in complete loss oftyrosine-hydroxylase stained neurons in the ipsilateral striatum, ascompared to preserved tyrosine-hydroxylase in the contralateral side(FIG. 18).

Human ES derived neural progenitors (as described in Example 1a) wereused for transplantation. The phenotype of cells within the neuralspheres was characterized prior to transplantation by immunocytochemicalstudies (as described in Example 1c) and RT-PCR.

For PT-PCR studies, total RNA was extracted from: (1) human ES cellcolonies (one week after passage), (2) free-floating spheres after 6weeks in culture, (3) differentiated cells growing from the spheres at 1week after plating on laminin in the presence of AA 400 μM (Sigma) andthe survival factors NT3 10 ng/ml, NT4 20 ng/ml and BDNF 10 ng/ml (allhuman recombinants from R&D). Total RNA was isolated using RNA STAT-60solution (TEL-TEST, Inc., Friendswood Tex.) or TRI-reagent (Sigma)followed by treatment with RNase-free DNase (Ambion, The RNA company,Austin Tex.). The cDNA synthesis was carried out using Moloney murineleukemia virus (M-MLV) reverse transcriptase and oligo (dT) as a primer,according to the manufacturers' instructions (Promega, Madison Wis.). Toanalyze relative expression of different mRNA, the amount of cDNA wasnormalized based on the signal from GAPDH mRNA. Levels of marker mRNAsexpressed by neural spheres and differentiated cells were compared tothat in the undifferentiated hES cells. PCR was carried out usingstandard protocols with Taq DNA Polymerase (Gibco invitrogencorporation). Amplification conditions were as follows: denaturation at94° C. for 15 seconds, annealing at 55-60 for 30 seconds, and extensionat 72° C. for 45 seconds. The number of cycles varied between 18 and 40,depending on the particular mRNA abundance. Primer sequences (forwardand reverse 5′-3′) and the length of amplified products were as follows:

(SEQ ID NO: 3) Oct4-CGTTCTCTTTGGAAAGGTGTTC, (SEQ ID NO: 4)ACACTCGGACCACGTCTTTC, 320 bp; (SEQ ID NO: 5) Otx2-CGCCTTACGCAGTCAATGGG,(SEQ ID NO: 6) CGGGAAGCTGGTGATGCATAG, 641 bp; (SEQ ID NO: 7)Pax2-TTTGTGAACGGCCGGCCCCTA, (SEQ ID NO: 8)CATTGTCACAGATGCCCTCGG, 300 bp; (SEQ ID NO: 9)Pax5-CCGAGCAGACCACAGAGTATTCA, (SEQ ID NO: 10)CAGTGACGGTCATAGGCAGTGG, 403 bp; (SEQ ID NO: 11)Lmx1B-TCCTGATGCGAGTCAACGAGTC, (SEQ ID NO: 12)CTGCCAGTGTCTCTCGGACCTT, 561 bp;  (SEQ ID NO: 13)Nurr1-GCACTTCGGCAGAGTTGAATGA, (SEQ ID NO: 14)GGTGGCTGTGTTGCTGGTAGTT, 491 bp; (SEQ ID NO: 15)En1-CTGGGTGTACTGCACACGTTAT, (SEQ ID NO: 16)TACTCGCTCTCGTCTTTGTCCT, 357 bp; (SEQ ID NO: 17)En2-GTGGGTCTACTGTACGCGCT, (SEQ ID NO: 18) CCTACTCGCTGTCCGACTTG, 368 bp;(SEQ ID NO: 19) AADC-CTCGGACCAAAGTGATCCAT, (SEQ ID NO: 20)GGGTGGCAACCATAAAGAAA, 252 bp; (SEQ ID NO: 21)TH-GTCCCCTGGTTCCCAAGAAAAGT, (SEQ ID NO: 22)TCCAGCTGGGGGATATTGTCTTC, 331 bp; (SEQ ID NO: 23)β-actin-CGCACCACTGGCATTGTCAT, (SEQ ID NO: 24)TTCTCCTTGATGTCACGCAC, 200 bp; (SEQ ID NO: 25)GAPDH-AGCCACATCGCTCAGACACC, (SEQ ID NO: 26) GTACTCAGCGCCAGCATCG 301 bp;Ptx3 (TGGGAGTCTGCCTGTTGCAG (SEQ ID NO: 27),CAGCGAACCGTCCTCTGGG (SEQ ID NO: 28) 372 bp)The hES Cell-Derived neural spheres were transplanted after partialmechanical dissociation of the spheres into small clumps into thestriatum of the rats (400,000 cells/animal) with a hamilton syringealong 2 tracts per striatum, using a stereotaxic device. Coordinate fortransplantation were A-P=0, L=3.5, H=−7.5 to −4.5 and A=1, L=2, H=−7.5to −4. Neural spheres that were passaged for 6 weeks (with highpotential of generating neurons and specifically dopaminergic neurons)and neural spheres that were passaged for 11 weeks (With a lowerpotential for generating dopaminergic cells) were transplanted. Controlrats underwent sham operation and were injected with saline. Animalsreceived Cyclosporin A treatment (10 mg/Kg) throughout the experiment.

At 2 weeks, 1 month, 2 months and 3 months after transplantation, theseverity of the disease was scored by pharmacological andnon-pharmacological tests and compared between hES cell transplanted andvehicle transplanted animals. Rotations were counted by a computerizedrotameter system for 1 hour after S.C. Injection of apomorphine (25μg/100 g body weight) and for 1 hour after I.P. d-amphetamine (4 mg/kgperformed 2 days later) (in early experiments, rotations were counted byan observer as described above). Non-pharmacological tests included thestepping adjustments (Olsson et al., 1995) and forelimb placing (Lindneret al., 1997) tests. The number of stepping adjustments was counted foreach forelimb during slow-sideway movements in forehand and backhanddirections over a standard flat surface. The stepping adjustments testwas repeated three times for each forelimb during three consecutivedays. The forelimb placing test assesses the rats' ability to makedirected forelimb movements in response to a sensory stimuli. Rats wereheld with their limbs hanging unsupported. They were then raised to theside of a table so that their whiskers made contact with the top surfacewhile the length of their body paralleled the edge of the tabletop.Normally, rats place their forelimb on the tabletop almost every time.Each test included ten trials of placing of each forelimb and wasrepeated in three consecutive days. The results of both tests areexpressed as percentage of forelimb stepping adjustments and placing inthe lesioned side compared to the non-lesioned side. The mean number ofrotations and the mean results (in percentage) of non-pharmacologicaltests were compared between the experimental groups using studentt-test.

The rats were then sacrificed and their brains processed forimmunohistochemical studies as previously described (Reubinoff et al.,2001) to determine the fate of the engrafted cells. Transplanted humancells were identified by immunohistochemistry for human specificmarkers, such as anti-human mitochondrial antibody and anti-humanribonucleic protein (Reubinoff et al 2001). Tyrosine-hydroxylase (TH)stains were performed on thick (20-40 micron) sections to quantify THdensity in the striatum and substantia nigra as well as on standard 8micron section to double stain and co-localize with the human specificmarkers.

A Rabbit anti TH antibody from Chemicon was used (at 1:100) followed bygoat anti-rabbit IgG secondary antibody, conjugated to Cy3 (Jacksonimmunoresearch laboratory PA; 1:500). TH density was quantified using acomputerized image analysis system. Graft survival was analyzed by thecomputerized image analysis system for staining of the human specificmarkers. Cell proliferation within the graft was analysed byimmunohistochemical evaluation of the percentage of cells that weredecorated with anti PCNA (Chemicon, mouse monoclonal; 1:100) or anti Ki67 (Novocastra Laboratories Ltd UK; rabbit polyclonal 1:100).Differentiation of transplanted cells into dopaminergic neurons wasconfirmed by immunohistochemical studies using anti human DAT (Chemicon;rat monoclonal 1:2000) followed by FITC conjugated goat anti rat(Molecular Probes)

Prior to implantation into Parkinsonian rats, the phenotype of the cellswithin spheres that were propagated 6 weeks in culture was characterizedas well as their developmental potential to give rise to midbrain DAneurons. Indirect immunofluorescence analysis following disaggregationof the spheres demonstrated that >90% of the cells within the spheresexpressed markers of neural progenitors (FIG. 19). Thus, as demonstratedabove, the sphere cultures were highly enriched for neural progenitors.

Successful differentiation of the hES cell derived NPs into midbrain DAneurons probably require the induction of the same key regulatory genesthat are expressed by neural progenitor cells during the development ofthe midbrain in vivo (Lee et al., 2000). Among these key genes are theOTX homebox genes (OTX1 and OTX2) that are widely expressed at the earlystages of neuroectoderm differentiation. Interactions between OTX genesare thought to specify the development of the midbrain and hindbrain(Simeone 1998; Acampora D and Simeone 1999). The genes Pax2, Pax5, Wnt1,En1 and En2 that are expressed further downstream during midbraindevelopment and serve as early organizers surrounding the ventralmidbrain progenitors neurons (Stoykova, & Gruss 1994; Rowitch & McMahon1995). Lastly, the transcription factors Nurr1 and Lmx1b that areimplicated in the final specification of the mesencephalic dopaminesystems (Zetterstrom et al., 1997; Smidt et al., 2000). These regulatorygenes were all expressed by the progenitors within the spheres. Thelevel of expression of these regulatory genes was up regulated upondifferentiation (FIG. 20). These findings suggested that the neuralprogenitors had the developmental potential to give rise to midbrain DAneurons. Some of the genes were also weakly expressed by cells withinthe hES cell cultures probably reflecting early background neuraldifferentiation. It should be noted that Oct4 was not expressed by thesphere cultures suggesting that the spheres did not includeundifferentiated hES cells.

In early experiments we have compared the rotational behaviour of alimited number of rats following transplantation of early passage andlate passage neural spheres.

In the experimental group that was transplanted with human neuralspheres that were passaged for 11 weeks prior to transplantation therewas a mild but statistically significant clinical effect at 3 monthspost-transplantation (FIG. 21). As compared to baselineapomorphine-induced rotational behavior, the control animals exhibited100.25±9% rotation, while the transplanted animals developed 79±19%rotations (p=0.04).

In the experimental group that was transplanted with human neuralspheres that were passaged for 6 weeks prior to transplantation, aclinical effect was observed as early as 1 month after transplantation.At this time point the rotational behavior in transplanted animals hadalready decreased to 72±14% of baseline (as compared to 102±18% in thecontrol group, p=0.04). Moreover, the effect of apomorphine in thetransplanted animals lasted a shorter length of time, with rotationalbehavior at 30 minutes after injection of apomorphine reducing to 65±12%of control (p=0.01). At 2 months after transplantation the rotationalbehavior, in transplanted rats decreased further to 58% of baseline(versus 102% in controls, p=0.006, student t-test). Again, theshortening of length of time of rotational behavior was evident, asafter 40 minutes, the transplanted animals exhibited 43% rotations ascompared to control (p=0.005, FIG. 22).

These results are in line with in-vitro studies and suggest that humanneural spheres (of early passage) that have a higher potential togenerate dopaminergic cells in-vitro also induce a stronger and morerapid clinical improvement in the Parkinsonian rats.

We have therefore transplanted in following experiments only earlypassage (6 weeks) spheres.

To further study the behaviour of Parkinsonian rats after stem celltransplantation we have extended the number of transplanted and controlanimals, included analysis of rotation after administration of bothapomorphine and amphetamine and evaluated the behaviour of animals innon-pharmacological tests. It should be noted that non-pharmacologicaltests provide a more direct measure of motor deficits analogous to thosefound in human Parkinson's disease (Kim et al., 2002). The results ofthe pharmacological tests are presented in FIGS. 23 and 24. These testsdemonstrated a significant reduction of rotational behaviour intransplanted animals. Both the stepping adjustments and forelimb placingnon-pharmacological tests also demonstrated a significant increase inmobility after stem cell therapy (FIG. 25). In conclusion, the resultsof both, the pharmacological and non-pharmacological tests, demonstrateda significant reduction of Parkinsonism following transplantation ofhuman ES cell-derived neural progenitors.

The fate of the transplanted human neural progenitors was studied byimmunohistochemistry and RT-PCR. The sites of transplants wereidentified on H&E stained coronal sections. The human cells were foundin the striatum along the injection tracts, identified byimmunostainings for human specific mitochondrial marker (FIG. 26), humanspecific ribonuclear protein and nestin (FIG. 27).

To evaluate the survival of the graft, sections were stained for humanmitochondria. We compared the size of transplants at 24 hours and 3months post transplantation. Since there was edema and free blood withinand around the transplants at 24 hours, we measured the amount of humancells by quantifying the human-specific mitochondrial staining inlow-power microscopic fields. This was calculated by multiplying theentire stained area with fluorescence intensity (above background). Atthe center of the transplant, the area of graft at 24 hours was 94+/−34(arbitrary units; n=6) and at 12 weeks it was 43+/−18 units (n=5). Thisindicates approximately 45% graft survival at 12 weeks aftertransplantation.

Given the potential of ES cells to generate teratomas aftertransplantation, we have evaluated the percentage of proliferating cellswithin the grafts. At 24 hours post transplantation, the majority ofcells (64.5%) were in a proliferative state as indicated by positivePCNA and ki67 staining. At 12 weeks, there were very rare (<0.2%)PCNA+cells (FIG. 28). Ki-67+ or PCNA+ cells were not observed in thehost striatal parenchyma near the graft. In addition, H&E stainedsections, covering the entire brain did not reveal teratomas or anyother tumor formation in transplanted rats.

Double staining with anti TH and anti human mitochondria at 12 weekspost-transplantation demonstrated that TH+ neurons were generated fromthe transplanted human cells (FIG. 29). The number of TH+ fibers countedin human mitochondria+ areas, relative to number of DAPI+ counterstainednuclei, indicated that 0.41/+0.3% of human cells generated TH+ fibers(n=9). If approximately 4×10⁵ viable cells were transplanted into eachrat, it may be estimated, therefore, that the transplants generated onaverage 740 TH+ neurons at 12 weeks post-transplantation.

To support the acquisition of a dopaminergic fate by the engrafted humanprogenitors, we have demonstrated by immunohistochemistry the expressionof human DAT within the lesioned striatum 12 weeks after transplantation(FIG. 29E). We have further demonstrated the expression of humanspecific transcripts of midbrain markers in brain samples fromtransplanted animals. Total RNA was extracted using the RNeasy kit(Qiagen) from midbrain samples that included the graft from stem cell(n=2) and vehicle transplanted (n=1) Parkinsonian rats. The RT-PCRreaction and the details of human specific primers are described above,Transcripts of human midbrain and dopaminergic neuron markers wereexpressed in samples from animals that received stem celltransplantation and were not detected in control animals (FIG. 30).

In conclusion the results of these experiments demonstrate the long-termsurvival of hES cell-derived neural progenitors after transplantation tothe stratum of parkinsonian rats. Proliferation of the transplantedcells decayed with time, teratoma tumor formation was not observed andthe engrafted progenitors differentiated in vivo into DA neurons thatled to functional recovery of Parkinsonism.

Example 4 Transplantation of Human Embryonic Stem Cell-Derived NeuralProgenitors Corrects Deficits in a Rat Parkinson Model

Highly enriched cultures of neural progenitors from hES cells weregrafted into the striatum of Parkinsonian rats. A significant fractionof the graft survived for at least 12 weeks, the transplanted cellsstopped proliferating and teratoma tumors were not observed. The graftedcells differentiated in vivo into DA neurons though at prevalence(0.41%) similar to the one observed following spontaneousdifferentiation in vitro. Transplanted rats exhibited significantimprovement in rotational behaviour that was induced by d-amphetamineand by apomorphine, and in stepping and placing non-pharmacologicalbehavioural tests. Long-term survival of the grafted cells, lack ofteratoma tumor formation, and the spontaneous differentiation of afraction of the transplanted cells into DA neurons that reduced motorasymmetries and improved behavioural deficits of Parkinsonian rats wasdemonstrated. This study indicates the potential of hES cells to inducefunctional recovery in an animal model of Parkinson's disease.

A. Development and Characterization of hES Cell-Derived NPs

Differentiation of hES cells into highly enriched cultures ofproliferating NPs was accomplished according to our simple two-stepprotocol (Reubinoff et al., 2001) with some modifications. In the firststep, hES cell colonies (FIG. 31A) were cultured for prolonged periodson feeders in the presence of the BMP antagonist noggin. Under theseculture conditions, the hES cells in most of the colonies differentiatedalmost uniformly into tightly packed small progenitor cells. Briefly,human ES cells (HES-1 cell line, Reubinoff et al. 2000) with a stablenormal (46XX) karyotype were cultured on mitomycin C treated mouseembryonic fibroblast feeder layer in gelatin-coated tissue culturedishes (FIG. 31A). To induce neural differentiation, clumps ofundifferentiated hES cells were plated on fresh mitotically inactivatedfeeders and cultured for eight days in serum containing medium comprisedof DMEM (Gibco, Gaithersburg, Md.), containing glucose 4500 mg/L withoutsodium pyruvate, supplemented with 10% fetal bovine serum (Hyclone,Logan, Utah), 0.1 mM beta-mercaptoethanol, 1% non-essential amino acids,2 mM glutamine, 50 u/ml penicillin, 50 μg/ml streptomycin (Gibco) and500 ng/ml noggin (R&D Systems Inc., Minneapolis, Minn.). The medium wasreplaced every other day. Noggin was then omitted and the cells werefurther cultured in the same medium for additional 6 days. At this time,70%-90% of the colonies differentiated almost uniformly into tightlypacked small cells with a uniform gray opaque appearance under darkfield stereomicroscopy (FIG. 31B). In parallel, the colonies acquired anearly uniform gray opaque appearance under dark field stereomicroscope(FIG. 3B).

In the second step, patches containing about 150 cells each were cut outfrom the gray opaque areas, using a razor blade (surgical blade #15),and replated in serum-free medium that consisted of DMEM/F12 (1:1), B27supplementation (1:50), glutamine 2 mM, penicillin 50 u/ml andstreptomycin 50 μg/ml (Gibco), and supplemented with 20 ng/ml humanrecombinant epidermal growth factor (EGF), and 20 ng/ml basic fibroblastgrowth factor (bFGF) (R & D Systems Inc.). The clusters of cellsdeveloped into round spheres that were sub-cultured once a week aspreviously described (Reubinoff et al., 2001) (FIG. 31C). The medium wasreplaced twice a week.

At this stage, prior to implantation into Parkinsonian rats, thephenotype of the cells within the spheres was characterized as well astheir developmental potential to give rise to midbrain DA neurons.Indirect immunofluorescence analysis following disaggregation of thespheres demonstrated that >90% of the cells within the spheres expressedmarkers of neural progenitors (FIG. 32J). Thus, the sphere cultures werehighly enriched for neural progenitors.

The regulatory genes (OTX1 AND OTX2, Pax2, Pax5, Wnt1, En1 and En2) andthe transcription factors Nurr1 and Lmx1b were all expressed by theprogenitors within the spheres suggesting that these neural progenitorshad the developmental potential to give rise to midbrain DA neurons(FIG. 32). Some of the genes were also weakly expressed by cells withinthe undifferentiated hES cell cultures.

The phenotype of the neural progenitors following spontaneousdifferentiation in vitro has been characterised. Upon withdrawal ofmitogens from the medium and plating on laminin, the spheres attachedrapidly, and cells migrated out to form a monolayer of differentiatedcells. The expression of transcripts of the regulatory genes of midbraindevelopment and markers of DA neurons was up regulated in thedifferentiated progeny (FIG. 32). After 7 days of differentiation,immunocytochemical studies were performed. Standard protocols were usedfor the immunophenotyping of disaggregated progenitor cells anddifferentiated cells following fixation with 4% paraformaldehyde.Primary antibodies localisation was performed by using swine anti-rabbitand goat anti-mouse immunoglobulins conjugated to fluoresceinisothiocyanate (FITC) (Dako, A/S Denmark; 1:20-50), goat anti mouse IgMconjugated to FITC (Jackson Lab. West Grove, Pa.: 1:100), goat antirabbit Ig conjugated to Texas Red (Jackson Lab, 1:100) and goat antimouse IgG conjugated to Cy^(TH)3 (Jackson Lab., 1:500). Proper controlsfor primary and secondary antibodies revealed neither non-specificstaining nor antibody cross reactivity.

To characterize the immunophenotype of cells within the aggregates,spheres that were cultivated for 6 weeks were mechanically partiallydisaggregated, and the resulting small clumps and single cells wereplated in serum-free medium, as described above, on poly-D-lysine (30-70kDa, 10 μg/ml, Sigma, St. Louis, Mo.) and laminin (4 μg/ml, Sigma). Thecells were fixed after 24 hours and examined for the expression of N-CAM(Dako; mouse IgG 1:10), nestin (rabbit antiserum a kind gift of Dr. RonMcKay; 1:25; or from Chemicon, Temecula, Calif.; rabbit anti human1:100-200), A2B5 (ATCC, Manassas, Va.; mouse clone 105 1:20), PSA-N-CAM(Developmental Studies Hybridoma Bank, Iowa city, Iowa; mouse undiluted,or from Chemicon Temecula, Calif. mouse 1:200). One-to-two hundred cellswere scored within random fields (at ×400) for the expression of each ofthese markers and the experiments were repeated at least 3 times.

To induce differentiation, spheres that were 6 weeks in culture, weredisaggregated into small clumps and plated on poly-D-lysine and lamininin serum-free growth medium (as described above) without supplementationof growth factors for 1 week. Differentiated cells were analysed for theexpression of GFAP (Dako; rabbit Ig 1:400), β-tubulin III (Sigma; mouseIgG 1:2000), serotonin (Sigma; rabbit 1:1000) and tyrosine hydroxylase(TH) (Pel-Freez anti Rabbit polyclonal, 1:100). To determine thepercentage of neurons, 200-500 cells were scored within random fields ofthe outgrowth from differentiating clumps (at ×400) and the experimentswere repeated at least 3 times.

From the immunocytochemical studies, it was found that 30% of the cellswere immunoreactive with anti β-tubulin III (a neuronal marker) (FIGS.31J, H and I). Double labelling studies showed that about 0.5% of thecells in these cultures co-expressed β-tubulin III and tyrosinehydroxylase (TH) (FIG. 31H), and 0.8-1% co-expressed β-tubulin III andserotonin (FIG. 31I). These results suggested that under our cultureconditions a low percentage (<11%) of the progenitors spontaneouslydifferentiated in vitro into putative mid/hind brain neurons.

B. Survival and Differentiation of Human nps After Transplantation toParkinsonian Rats

The survival, differentiation and function of the hES cell-derived NPsin vivo after transplantation to the rat animal model of Parkinson'sdisease was analysed.

A Parkinson's disease model was induced in adult Sprague-Dawley rats bystereotaxic injection of the neurotoxin 6-hydroxydopamine to causeunilateral nigrostriatal lesions. 8 μg/rat of 6-hydroxydopamine wereInjected in 4 μl into the right Substantia Nigra. 6-hydroxydopamine wasinjected to the right substantia-nigra to deplete dopaminergicinnervation in the ipsilateral stratum. Coordinates of injection wereP=4.8, L=1.7, H=−8.6. Preliminary experiments confirmed this resulted inthe complete loss of TH+stained neurons in the ipsilateral striatum,whereas TH expression in the contra-lateral side was preserved.

At 18 days after the lesion, Parkinsonian rats with >350 rotations perhour after S.C. injection of apomorphine (25 mg/100 gr body weight) wereselected for the transplantation experiment.

At 3 weeks after the lesion, hES-cell derived neural spheres that hadbeen passaged for 6 weeks were grafted (along two tracts, 4×10⁵cells/animal) into the right striatum of rats that were preselected forapomorphine-induced high rotational activity. Two days after selectionof Parkinsonian rats, animals were stereotaxically injected with eitherneurospheres or medium into 2 sites of the right striatum. ThehES-derived neural spheres (passaged for 6 weeks) were mechanicallydissociated into small clumps and transplanted (about 400,000 cells in12-14 μl/animal) with a hamilton syringe, along 2 tracts per striatum,using a stereotaxic device. Coordinates for transplantation were:anteromedial tract—A−P=0, L=3.5, H=−7.5 to −4.5 and posterolateraltract—A=1, L=2, H=−7.5 to −4. Control rats underwent sham operation andwere injected with vehicle solution. To prevent rejection of graftedhuman cells, all rats (transplanted and controls) received daily I.Pinjection of 10 mg/kg cyclosporine A (Sandimmune, Sandoz).

The rats were sacrificed for histopathological analysis of the graft 24hours after transplantation (n=6), and after behavioral follow up of 12weeks (21 sphere and 17 vehicle grafted rats).

At the end of follow-up and behavioral studies, rats were euthanized bypentobarbital overdose and perfused with saline and 4% paraformaldehyde.Serial 8 82 m coronal frozen sections were prepared and every seventhsection was stained with hematoxylin and eosin (H&E) to identify thegraft in the brain. In areas in which a graft was identified, thesections were post fixated with 4% formaldehyde. Immunofluorescentstainings were performed with the following primary antibodies;human-specific mitochondrial antibody (mouse IgG, Chemicon; 1:20),tyrosine hydroxylase (TH rabbit IgG, Chemicon 1:100), nestin (antibodyas detailed above; 1:50), neurofilament heavy chain (NF-200 mouse IgG,Sigma; 1:100), β III-tubulin (antibody as detailed above; 1:50),neuronal nuclei marker (NeuN mouse IgG Chemicon 1:50).

Sections that were post fixated with acetone were stained withhuman-specific ribonuclear protein antibody (RNP, mouse IgM, Chemicon;1:20). Sections that were post fixated with methanol were stained withProliferating Cell Nuclear Antigen (PCNA mouse IgG Chemicon 1:100), Ki67Antigen (Rabbit polyclonal, Novocastra laboratories 1:100), vesicularmonoamine transporter 2 (VMAT2, rabbit Pel-Freez 1:50-100), humandopamine transporter (rat monoclonal, Chemicon 1:2000). Goat anti mouseIgG conjugated to Alexa 488 or Cy3, goat anti mouse IgM conjugated totexas red, goat anti rabbit IgG conjugated to Alexa 488 or to texas red(Jackson; 1:100) and goat anti rat IgG conjugated to Alexa 488(Molecular Probes 1:500) were used where appropriate for detection ofprimary antibodies. Double stains were performed by using primaryantibodies of different species or Ig subtype, followed bynon-cross-reactive secondary antibodies. Double labeling for TH andhuman mitochondria was used to evaluate the percentage of TH+ neuronswithin the grafts. At least 3 high power microscopic fields per sectionand 3 sections per animal were counted for TH+cells within the graft.Images were taken by a fluorescent microscope (Nikon E600) or confocalmicroscope (Zeiss), using channels for Alexa 488 fluorescence, Cy3 andCy5 fluorescence and Nomarsky optics.

The grafts were easily identified on H&E stained sections and byfluorescent DAPI nuclear counterstaining. At 12 weeks aftertransplantation, a graft was found in 17 animals. In each of theseanimals, two grafts were found, most often as a tubular mass of cellsalong the needle tract within the striatum. In five animals one of thetwo grafts was ectopic and was observed as a round mass in the cortex.

Anti human-specific mitochondria antibodies to specifically identifyhuman cells in transplanted rat brain sections (FIG. 33A) were used.Identification of human cells was confirmed by staining forhuman-specific ribo-nuclear protein (FIG. 33B). At 24 hourspost-transplantation, there was widespread expression of nestin in thegraft (FIG. 33C). At 12 weeks post-transplantation, the positive humanmitochondria cells were found only at the site of transplantation, andthere was no indication for cell migration to neighboring regions.

To evaluate graft survival, estimations of the number of transplantedcells at 24 hours and 3 months post-transplantation were compared. Sinceactual counting of the grafted cells was not practical an approach thatallowed rough estimation and comparison of the number of cells withinthe grafts was used. A coronal section along and through the center ofthe tubular transplants was identified and chosen from serial H&Estained coronal sections. Assuming that the graft had a symmetricaltubular structure, the area of the graft in the section was proportionalto the volume of the graft. Since there was edema and some free bloodwithin and around the transplants at 24 hours, and the density of humancells differed between grafts, the area of the grafts in the selectedcoronal sections was not representative of the number of cells. Toovercome this problem an adjacent section was stained for humanmitochondria and the amount of human cells by quantifying thehuman-specific mitochondrial staining was measured. This was calculatedby multiplying the entire stained area (in low-power microscopic fields)with fluorescence intensity above background. Twenty-four hours aftertransplantation, the overall graft mitochondrial staining in sectionsthrough the center of the transplants was 94+/−34 (arbitrary units; n=6)and at 12 weeks it was 43+/−18 units (n=5). This indicated approximately45% graft survival at 12 weeks after transplantation.

Double staining with human specific markers and neuronal markers,indicated the generation of human mitochondria+, neurofilament+(FIG.33D) and human ribonuclear protein+, Neun+ (FIG. 33E) neurons fromtransplanted cells. Double staining for human mitochondria andtyrosine-hydroxylase (TH) showed the presence of graft-derived TH+ cellsand fibers (FIG. 33F-H). The vehicle-grafted animals showed no THstaining in the ipsilateral substantia nigra or the striatum. At 12weeks post-transplantation, the number of TH+ cells in the humanmitochondria stained areas, relative to the number of DAPI+counterstained nuclei, indicated that 0.41+/−0.3% of the human cellsgenerated TH+ neurons (n=9 animals). Since 4×10⁵ cells were transplantedinto each rat, it may be estimated, therefore, that the graft generatedapproximately 740 TH+ neurons.

Cells that were decorated with an antibody directed against humandopamine transporter were identified within the graft and wereundetectable in the striatum of medium-grafted controls (FIG. 33I). Inaddition, an antibody directed against human vesicular monoaminetransporter (V-MAT2), a dopaminergic neuronal marker (Miller et al.,1999), decorated graft-derived cells that co-labeled with the human antimitochondrial antibody (FIG. 33J,K). V-MAT2+ cells were not observed inthe striatum of sham operated animals.

To confirm the expression of human dopaminergic neuronal markers in thetransplanted brains, RT-PCR analysis of striatal samples from vehicleand neural progenitor grafted rats was performed (as in Example 3).Transcripts of human midbrain and dopaminergic neuron markers wereexpressed in samples from animals that received stem celltransplantation (n=3) and were not detected in vehicle-transplantedanimals (n=2). The expression of these human-specific mRNA transcriptswas found only in the transplanted side, and not in the non-lesionedside of the same animals (FIG. 34).

Given the potential of ES cells to generate teratomas aftertransplantation, we evaluated the percentage of proliferating cellswithin the grafts. At 24 hours post-transplantation, the majority ofcells (64.5%) were in a proliferative state as indicated by positivePCNA (FIG. 33L) and ki67 staining (not shown). At 12 weeks, there werevery rare (<0.2%) PCNA+ cells (FIG. 33M). In addition, H&E stainedsections covering the entire brain, did not reveal teratomas or anyother tumor formation in transplanted rats.

C. Functional Recovery in Parkinsonian Rats After Transplantation ofhES-Derived Neural Spheres

At 2 weeks, 1 month, 2 months, and 3 months after transplantation, theseverity of the disease was scored by pharmacological tests and comparedbetween hES cell transplanted and vehicle transplanted animals.Rotations were counted for 1 hour after S.C. injection of apomorphine(25 μg/100 g body weight) and for 1 hour after I.P. d-amphetamine (4mg/kg, performed 2 days later) by a computerized rotometer system(San-Diego Instruments, Inc).

Pharmacological-induced rotational behavior was measured in rats thatwere transplanted with spheres or with medium at 2 weeks (Baseline), 4weeks, 8 weeks and 12 weeks after engraftment (FIG. 35). The 2 rats inwhich no graft was found did not exhibit any improvement in motorfunction. In transplanted rats (n=10 rats in which a graft was found),amphetamine-induced rotations decreased from 607±200/hour at baseline to334±130/hour at 12 weeks (45% decrease, p=0.001, FIG. 35A). It should benoted that the difference was already statistically significant at 8weeks. Amphetamine-induced rotations increased in the control group(n=10 rats) from 48±210/hour to 571±235/hour at 12 weeks.Apomorphine-induced rotations decreased in the transplanted group (n=19rats) from an average of 624±220/hour at baseline to 423±158rotations/hour at 12 weeks (31% decrease, p=0.0015, FIG. 35B). Thedifference between the groups was significant already at 8 weeks. Thecontrol group (n=17) rotated 567±169 times per hour at baseline and571±112 after 12 weeks.

Non-pharmacological tests were performed at 2 weeks and 3 months aftertransplantation. These included stepping adjustments (Olsson et al.,1995) and forelimb placing (Lindner et al., 1997) tests. The number ofstepping adjustments was counted for each forelimb during slow-sidewaymovements in forehand and backhand directions over a standard flatsurface. The stepping adjustments test was repeated three times for eachforelimb during three consecutive days. The forelimb-placing testassesses the rats' ability to make directed forelimb movements inresponse to a sensory stimulus. Rats were held with their limbs hangingunsupported. They were then raised to the side of a table so that theirwhiskers made contact with the top surface while the length of theirbody paralleled the edge of the tabletop. Normally, rats place theirforelimb on the tabletop almost every time. Each test included tentrials of placing of each forelimb and was repeated in three consecutivedays. The results of both tests are expressed as percentage of forelimbstepping adjustments and placing in the lesioned side as compared to thenon-lesioned side. The mean number of rotations and the mean results (inpercentage) of non-pharmacological tests were compared between theexperimental groups using the student t-test.

The behavioral analysis was extended to include also the steppingadjustments (Olsson et al., 1995) and forelimb placing (Lindner et al1997) non-pharmacological tests. Non-pharmacological tests provide amore direct measure of motor deficits analogous to those found in humanParkinson's disease (Kim et al., 2002). Stepping and placing wereexamined at baseline (2 weeks) and at 12 weeks after transplantation. At2 weeks the transplanted rats did not make any stepping or placing inthe lesioned side. At 12 weeks there was a significant improvement inboth non-pharmacological tests as compared to baseline and to controlrats (FIG. 35 C, D).

Values in the behavioural tests are given as mean±standard error.Statistical analysis for the pharmacological tests was performed byone-tailed analysis of variance (ANOVA), followed by Bonferroni post hoctest. In the non-pharmacological tests the groups were compared by thestudent's t-test. A statistical significant difference was consideredwhen p<0.05.

This study shows that transplantation of hES cell-derived neural spheresimproves the motor function in rats in an experimental model ofParkinson's disease.

A simple two-step protocol to direct the differentiation of hES cellsin-vitro into highly enriched cultures of proliferating NPs has beenused. The NPs expressed transcripts of key regulatory genes of midbraindevelopment as well as markers of dopaminergic neurons supporting theirpotential to differentiate into midbrain dopaminergic neurons. Followingtransplantation, a significant clinical effect was evident by all fourdifferent behavioral tests. Functional recovery in the pharmacologicalbehavioral tests was evident and significant at 8 and 12 weeks aftertransplantation. The functional recovery in the lesioned rats wascorrelated by the demonstration of transplant-derived dopaminergiccells, as indicated by immunofluorescent stainings and RT-PCR. At theRNA level, human specific transcripts of key regulatory genes ofmidbrain development as well as markers of DA neurons were observed inbrain samples from the location of the graft. At be protein level, humancells decorated with antibodies against dopamine neuron specific markersincluding DAT (Kim et al 2002) and VMAT2 (Miller et al., 1999) wereobserved within the grafts. Collectively this data suggest that thebehavioural recovery that we have observed Was related to thedifferentiation of the grafted NPs into functional DA neurons. However,further studies are required to confirm that hES cells can differentiateinto DA neurons with phenotype, function and interaction with hostneurons that are identical to those of authentic midbrain DA neurons.

These findings suggest that commitment of the human cells to adopaminergic fate prior to their transplantation is a pre-requisite toobtain a larger number of graft-derived dopaminergic neurons.

Transplantation of low dose of undifferentiated mouse ES cells intoparkinsonian rats resulted in the formation of teratomas in a highpercentage of animals (Bjorklund et. al., 2002). Here, the human EScells were directed to differentiate in-vitro into neural progenitorsprior to transplantation and ceased to express the transcription factorOct-4, a marker of undifferentiated ES cells. The human grafts did notproduce teratomas or non-neural tissue in the rat brains. Incorrelation, the transplanted cells ceased to express markers ofproliferating cells. Nevertheless, additional extensive long-termstudies are required to determine the safety of human ES derived neuralprogeny transplantation and to rule out potential hazards such as tumorformation or the development of cells from other lineages.

This study shows for the first time that human ES cell-derived NPs caninduce functional recovery in an experimental model of Parkinson'sdisease. The therapeutic effect, demonstrated in this study, indicatethe potential of hES cells for transplantation therapy and encouragefurther efforts that may eventually allow the use of hES cells for thetreatment of neurological disorders.

REFERENCES

Acampora D and Simeone A. Understanding the roles of Otx1 and Otx2 inthe control of brain morphogenesis. Trends Neuroscience 1999; 22:116-122.

Ben-Hur T, Einstein O, Mizrachi-Kol R, Ben-Menachem O, Reinhartz E,Karussis D, Abramsky O. Transplanted multipotential neural precursorcells migrate into the inflamed white matter in response to experimentalautoimmune encephalomyelitis. Glia 2003; 41: 73-80.

Bjorklund A and Lindvall O. Cell replacement therapies for centralnervous system disorders. Nature Neuroscience 2000; 3: 537-544.

Bjorklund L M, Sanchez-Pernaute R, Chung S, Andersson T, Chen I YMcNaught K S, Brownell A L, Jenkins B G, Wahlestedt C, Kim K S, IsacsonO. Embryonic stem cells develop into functional dopaminergic neuronsafter transplantation in a Parkinson rat model. Proc Nati Acacd Sci USA.2002 Feb. 19; 99(4):2344-9.

Freed C R et al. Transplantation of embryonic dopamine neurons forsevere Parkinson's disease. N Engl J Med 2001; 344: 710-9.

Gropp M, Itsykson P, Singer O, Ben-Hur T, Reinhartz E, Galun E,Reubinoff B E. Stable genetic modification of human embryonic stem cellsby lentiviral vectors. Molecular Therapy 2003; 7: 281-287.

Heikinheimo M, Lawshe A, Shackleford G M, Wilson D B, NacArthur C A.FGF-8 expression in the post gastrulation mouse suggests roles in thedevelopment of the face, limbs, and central nervous system. Mach Dev1994; 48: 129-38.

Hoshikawa M, Ohbayashi N, Yonamine A, Konishi M, Ozaki K, Fukui S, ItohN. Structure and expression of a novel fibroblast growth factor, FGF-17,preferentially expressed in the embryonic brain. Biochem Biophys ResCommun 1998 Mar. 6; 244.

Isacson O and Deacon T. Neural transplantation studies reveal the brainscapacity for continuous reconstruction. Trends Neurosci 1997; 477-82.

Kawasaki H et al. Induction of midbrain dopaminergic neurons from EScells by stromal cell derived inducing activity. Neuron 2000; 28: 31-40.

Kim J H et al. Dopamine neurons derived from embryonic stem cellsfunction in an animal model of Parkinson's disease. Nature 2002; 418:50-6.

Lee S H et al. Efficient generation of midbrain and hindbrain neuronsfrom mouse embryonic stem cells. Nature Biotechnology 2000; 18: 675-679.

Lindvall O. Neural transplantation: a hope for patients with Parkinson'sdisease. Neuroreport 1997; 8(14): iii-x.

Lindner M D et al. Rats with partial striatal dopamine depletionsexhibit robust and long-lasting behavioral deficits in a simplefixed-ratio bar-pressing task. Behav Brain Res 1997; 86: 25-40.

Miller G W, Erickson J D, Perez J T, Penland S N, Mash D C, Rye D B,Levey A I. Immunochemical analysis of vesicular monoamine transporter(VMAT2) protein in Parkinson's disease. Exp Neurol. 1999; 156:138-48.

Olsson M et al. Forelimb akinesia in the rat Parkinson model:differential effects of dopamine agonists and nigral transplants asassessed by a new stepping test. J Neurosci 1995; 15: 3863-75.

Pera M F et al. Human embryonic stem cells. J Cell Sci. 2000; 113: 5-10.Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani C, Dina G, GalliR, Del Carro U, Amadio S, Bergami A, Furlan R, Comi G, Vesovi A L,Martino G. injection of adult neurospheres induces recovery in a chronicmodel of multiple sclerosis. Nature 2003; 422: 688-94

Reubinoff B E, Pera M F, Fong C Y, Trounson A, Bongso A. Embryonic stemcell lines from human blastocysts: somatic differentiation in vitro. NatBiotechnol 2000; 18(4):399-404.

Reubinoff B E, Itsykson P, Turetsky T, Pera M F, Reinhart E, Itzik A,Ben-Hur T. Neural progenitors from human embryonic stem cells. NatBiotechnol 2001 December; 19(12):1134-40

Rowitch D H & McMahon A P. Pax-2 expression in the murine neural plateprecedes and encompasses the expression domains of Wnt-1 and En-1. MechDev 1995; 52: 3-8.

Sanchez-Pernaute R, Studer L, Bankiewicz K S, Major E O, and McKay R D.In vitro generation and transplantation of precursors-derived humandopamine neurons. J Neurosci. Res. 2001; 65: 284-288.

Simeone A. Otx1 and Otx2 in the development and evolution of themammalian brain. EMBO 1998; 17: 6790-6798.

Smidt M P et al. A second independent pathway for development ofmesencephalic dopaminergic neurons requires Lmx1b. Nat Neurosci 2000; 3:337-41.

Stoykova A & Gruss P. Roles of Pax genes in developing and adult brainas suggested by expression patterns. J Neuroscience 1994; 14:1395-1412.

Studer L, Tabar V, McKay R D. Transplantation of expanded mesencephalicprecursors leads to recovery in parkinsonian rats. Nature Neuroscience1998; 1: 290-295.

Stull N D & Lacovitti L. Sonic hedgehog and FGF8: inadequate signals forthe differentiation of a dopamine phenotype in mouse and human neuronsin culture. Exp Neurol. 2001; 169: 36-43.

Xu J, Lawshe A, MacArthur G A, Ornitz D M. Genomic structure, mapping,activity and expression of FGF-17. Mech Dev 1999; 83: 165-78.

Xu J, Liu Z, Ornitz D M. Temporal and spatial gradients of FGF-8 andFGF-17 regulate proliferation and differentiation of midline cerebellarstructures. Development 2000; 127: 1833-43.

Zetterstrom R H et al. Dopamine neuron agenesis in Nurr1-deficient mice.Science 1997; 276: 248-50

Finally it is to be understood that various other modifications and/oralterations may be made without departing from the spirit of the presentinvention as outlined herein.

What is claimed is:
 1. A method of generating a tyrosine-hydroxylase positive (TH⁺) cell, said method comprising: (a) culturing a human embryonic stem (hES) cell with a BMP antagonist and FGF-2 to obtain a neural progenitor cell (NPC) in a neuroprogenitor medium; and subsequently (b) culturing the neural progenitor cell in a medium comprising a Fibroblast Growth Factor (FGF) selected from the group consisting of FGF-1, FGF-8 and FGF-17 or any combination thereof, thereby generating a tyrosine-hydroxylase positive (TH⁺) cell.
 2. The method of claim 1, wherein said BMP antagonist comprises noggin.
 3. The method of claim 1, wherein said medium further comprises at least one compound selected from the group consisting of Sonic Hedgehog Protein (SHH), cAMP inducers, Protein Kinase C (PKC) inducers and dopamine or any combination thereof.
 4. The method of claim 1, wherein the FGF is selected from FGF-1 and FGF-17.
 5. The method of claim 4 further comprising FGF-8.
 6. The method of claim 1, wherein said neurogrogenitor medium further comprises EGF and/or LIF.
 7. The method of claim 6, wherein said EGF is approximately 20 ng/ml and said LIF is approximately 10 ng/ml.
 8. The method of claim 1, wherein said FGF-1, said FGF-8 and said FGF-17 is present at a concentration of approximately 100 to 200 ng/ml.
 9. The method of claim 3, wherein said SHH is present at a concentration of about 0.5 to 1 μg/ml.
 10. The method of claim 3, wherein said cAMP inducer is present at a concentration of 50 μm to 0.25 mm.
 11. The method of claim 3, wherein said PKC inducer is present at a concentration of approximately 20 μm.
 12. A method according to claim 1 wherein the neural progenitor cell is cultured in the presence of neural fate inducers present in any one of the following combinations: (i) FGF-1, FGF-8, FGF-17, SHH, IBMX, forskolin, PMA/TPA and dopamine; or (ii) FGF-8, SHH, IBMX, forskolin, PMA/TPA and dopamine; or (iii) FGF-17, SHH, IBMX, forskolin, PMA/TPA and dopamine; or (iv) FGF-1, IBMX, forskolin, PMA/TPA and dopamine.
 13. The method of claim 1, wherein the medium further comprises ascorbic acid (AA) or an analogue thereof.
 14. The method of claim 1, further including culturing the neural progenitor cell in the presence of NT4 or an equivalent thereof.
 15. The method of claim 2, wherein said noggin is used at a concentration of 350-700 ng/ml.
 16. A composition for generating a tyrosine-hydroxylase positive (TH⁺) cell in a cultured hES cell, said composition comprising a neural fate inducer in any one of the following combinations: (i) FGF-1, FGF-8, FGF-17, SHH, IBMX, forskolin, PMA/TPA and dopamine; or (ii) FGF-8, SHH, IBMX, forskolin, PMA/TPA and dopamine; or (iii) FGF-17, SHH, IBMX, forskolin, PMA/TPA and dopamine; or (iv) FGF-1, IBMX, forskolin, PMA/TPA and dopamine. 